Severe acute respiratory syndrome coronavirus

ABSTRACT

An outbreak of a virulent respiratory virus, now known as Severe Acute Respiratory Syndrome (SARS), was identified in Hong Kong, China and a growing number of countries around the world in 2003. The invention relates to nucleic acids and proteins from the SARS coronavirus. These nucleic acids and proteins can be used in the preparation and manufacture of vaccine formulations, diagnostic reagents, kits, etc. The invention also provides methods for treating SARS by administering small molecule antiviral compounds, as well as methods of identifying potent small molecules for the treatment of SARS.

All documents cited herein are incorporated by reference in their entirety.

RELATED APPLICATIONS, FROM WHICH PRIORITY IS CLAIMED

This application incorporates by reference in its entirety U.S. provisional patent application 60/462,218, Attorney Reference No. PP20474.001, filed on Apr. 10, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/462,465, Attorney Reference No. PP20480.001, filed on Apr. 11, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/462,418, Attorney Reference No. PP20480.002, filed on Apr. 12, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/462,748, Attorney Reference No. PP20480.003, filed on Apr. 13, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/463,109, Attorney Reference No. PP20480.004, filed on Apr. 14, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/463,460, Attorney Reference No. PP20480.005, filed on Apr. 15, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/463,668, Attorney Reference No. PP20480.006, filed on Apr. 16, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/463,983, Attorney Reference No. PP20480.007, filed on Apr. 17, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/463,971, Attorney Reference No. PP20480.008, filed on Apr. 18, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/464,899, Attorney Reference No. PP20480.009, filed on Apr. 22, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/464,838, Attorney Reference No. PP20507.001, filed on Apr. 22, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/465,273, Attorney Reference No. PP20518.001, filed on Apr. 23, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/465,535, Attorney Reference No. PP20518.002, filed on Apr. 24, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/468,312, Attorney Reference No. PP20480.010, filed on May 5, 2003 via Express Mail with the US post office, and U.S. provisional patent application 60/473,144, Attorney Reference No. PP20480.011, filed on May 22, 2003, U.S. provisional patent application 60/495,024, Attorney Reference No. PP20480.012, filed on Aug. 14, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/505,652, Attorney Reference No. PP20480.013, filed on Sep. 23, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/510,781, Attorney Reference No. PP20480.014, filed on Oct. 11, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/529,464, Attorney Reference No. PP20480.015, filed on Dec. 11, 2003 via Express Mail with the US post office, U.S. provisional patent application 60/536,177, Attorney Reference No. PP20480.016, filed on Jan. 12, 2004 via Express Mail with the US post office, and U.S. provisional patent application 60/______, Attorney Reference No. PP20480.017, filed on Apr. 7, 2004 via Express Mail with the US post office.

FIELD OF THE INVENTION

The invention relates to nucleic acids and proteins from Severe Acute Respiratory Syndrome (SARS) Virus. These nucleic acids and proteins can be used in the preparation and manufacture of vaccine formulations for the treatment or prevention of SARS. The invention also relates to diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention also relates to methods for the treatment or prevention of SARS utilizing small molecule viral inhibitors and combinations of small molecule viral inhibitors and kits for the treament of SARS.

BACKGROUND OF THE INVENTION

An outbreak of a virulent respiratory virus, now known as Severe Acute Respiratory Syndrome (SARS), was identified in Hong Kong, China and a number of other countries around the world in 2003. Patients typically had symptoms including fever, dry cough, dyspnea, headache, and hypoxemia. Isolates of the SARS virus appear to have homology with at least the RNA polymerase gene of several known coronaviruses. A phylogenetic analysis of this homology is presented in Peiris et al., “Coronavirus as a possible cause of severe acute respiratory syndrome”, Lancet, published online Apr. 8, 2003 at http://image.thelancet.com/extras/03art3477web.pdf, incorporated herein by reference in its entirety. Other sequenced fragments of the SARS virus genome appear to overlap with the open reading frame 1b of coronaviruses. See, Drosten et al., “Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome”, New England Journal of Medicine, published online at http://www.nejm.org on Apr. 10, 2003, incorporated herein by reference in its entirety.

The Genome Science Center in British Colombia, Canada published on its website (http://www.bcgsc.ca/bioinfo/SARS/) a draft genome assembly of 29,736 base pairs of a virus believed to be a SARS virus, referred to as the TOR2 isolate. This draft genome assembly is given herein as SEQ ID NO: 1.

The Centers for Disease Control (CDC) published a nucleotide sequence of a SARS-CoV strain (SEQ ID NO: 2) on its website (http://www.cdc.gov/ncidod/sars/pdf/nucleoseq.pdj). The CDC has also published a phylogenetic tree of the predicted N, S and M proteins (attached as FIG. 6). This tree places the SARS virus outside any of the previously known coronavirus groups.

There is a growing need for prophylactic or therapeutic vaccines against the SARS virus as well as diagnostic and screening methods and compositions to identify the presence of the virus in, e.g., mammalian tissue or serum.

SUMMARY OF THE INVENTION

The invention relates to nucleic acids and proteins from Severe Acute Respiratory Syndrome (SARS) virus. These nucleic acids and proteins can be used in the preparation and manufacture of vaccine formulations for the treatment or prevention of SARS. Such vaccine formulations may include an inactivated (or killed) SARS virus, an attenuated SARS virus, a split SARS virus preparation and a recombinant or purified subunit formulation of one or more SARS viral antigens. Expression and delivery of the polynucleotides of the invention may be facilitated via viral vectors and/or viral particles.

The invention also relates to diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention further includes non-coding SARS viral polynucleotide sequences, SARS viral sequences encoding for non-immunogenic proteins, conserved and variant SARS viral polynucleotide sequences for use in such diagnostic compositions and methods.

The invention further relates to vaccine formulations comprising one or more SARS virus antigens and one or more other respiratory virus antigens. Additional respiratory virus antigens suitable for use in the invention include antigens from influenza virus, human rhinovirus (HRV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), adenovirus, metapneumovirus, and rhinovirus. The additional respiratory virus antigen could also be from a coronavirus other than the SARS coronavirus. Preferably, the additional respiratory virus antigen is an influenza viral antigen.

The compositions of the invention may further comprise one or more adjuvants. Adjuvants suitable for use in the invention include mucosal, transdermal or parenteral adjuvants. Mucosal adjuvants suitable for use in the invention include detoxified bacterial ADP-ribosylating toxins, such as E. coli heat labile toxoids (e.g., LTK63), chitosan and derivatives thereof, and non-toxic double mutant forms of Bordetella pertussis toxoids. Parenteral adjuvants suitable for use in the invention include MF59 and aluminum or aluminum salts.

The invention also provides methods for treating SARS by administering small molecule compounds, as well as methods of identifying potent small molecules for the treatment of SARS.

In one aspect of the invention a method of identifying a therapeutically active agent is provided comprising: (a) contacting the therapeutically active agent with a cell infected with the SARS virus; (b) measuring attenuation of a SARS related enzyme.

In a more particular embodiment, the therapeutically active agent is a small molecule. In another more particular embodiment, the therapeutically active agent is a nucleoside analog. In another more particular embodiment the therapeutically active agent is a peptoid, oligopeptide, or polypeptide. In another embodiment the SARS related enzyme is SARS protease. In another embodiment the SARS related enzyme is SARS polymerase. In still another embodiment the SARS related enzyme is a kinase. Methods of identifying therapeutically active agents for treatment of SARS virus infection are further discussed in Section V below.

In another aspect of the invention a method of treating a human infected with SARS is provided comprising administering a small molecule to a patient in need thereof. In one embodiment the small molecule is an inhibitor of SARS protease. In another embodiment the small molecule is an inhibitor of SARS polymerase. In another embodiment the SARS related enzyme is a kinase. In still another embodiment the small molecule is administered orally or parenterally.

The invention also provides the use of such small molecules in the manufacture of a medicament for the treatment of severe acute respiratory syndrome.

Small molecule compounds of the present invention include those of less than 1000 g/mol, preferably with an aromatic region and greater than one heteroatom selected from O, S, or N. Preferred small molecules include, but are not limited to acyclovir, gancyclovir, vidarabidine, foscamet, cidofovir, amantidine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, and combinations thereof. Interferons may also be used for treating patients, including interferon-α and interferon-β. Interferon treatment has shown promise in treating SARS in monkeys (Enserink (2004) Science 303:1273-1275), particularly when pegylated (Haagmans et al. (2004) Nature Medicine 10:290-293).

One aspect of the present invention relates to methods for identifying individuals exposed to, and biological samples containing SARS virus (SARSV), and to kits for carrying out the methods. Such methods can utilize nucleic acid detection techniques such as PCR, RT-PCR (the Coronaviridae are RNA viruses), transcription-mediated amplification (TMA), ligase chain reaction (LCR), branched DNA signal amplification assays, isothermal nucleic acid sequence based amplification (NASBA), other self-sustained sequence replication assays, boomerang DNA amplification, strand-displacement activation, cycling probe technology, or combinations of such amplification methods. Such nucleic acid detection techniques utilize oligonucleotides having nucleotide sequence similar to, or complementary to, the SARS viral genome, as primers (e.g., for amplification) and as probes (e.g., for capture or detection), as is well known in the art.

Alternatively, or in addition to the nucleic acid detection methods described supra, the methods of the present invention can utilize various immunoassay techniques for detection of SARSV antigens and/or antibodies.

Accordingly, the present invention relates to methods of identifying individuals exposed to SARSV, or biological samples containing SARSV, by detecting the presence of SARSV antigens using antibodies which specifically bind to the same. The antibodies are preferably monoclonal antibodies. Quantification of the amount of viral antigens present in a sample of an individual may be used in determining the prognosis of an infected individual. Preferably, the SARSV antigens to be detected are generally one of the structural proteins, particularly those present on the surface of the viral particles and include, for example, the spike glycoprotein (S), also called E2; the envelope (small membrane) protein (E), also called sM; the membrane glycoprotein (M), also called E1 ; the hemagglutinin-esterase glycoprotein (HE); also called E3; and the nucleocapsid phosphoprotein (N). In preferred embodiments, the antigens to be detected are the S, E and M proteins using antibodies to the same.

The present invention relates to kits for identifying individual SARSV and reagents used in such kits. The kits comprise a first container which contains antibodies which specifically bind to a SARSV antigen and a second container which contains the SARSV antigen. The antibodies are preferably monoclonal antibodies. The kits may be adapted for quantifying the amount of antigen in a sample of an individual. Such information may be used in determining the prognosis of an infected individual.

The present invention relates to methods of identifying individuals exposed to SARS virus, or biological samples containing SARSV, by detecting the presence of antibodies against SARS virus antigen in a sample using SARS antigen. Quantification of the amount of anti-SARS protein from SARS antibodies present in a sample of an individual may be used in determining the prognosis of an infected individual. Any one or more of the viral proteins (structural proteins or nonstructural proteins) may be used as antigen to detect the SARSV antibodies; preferably a SARSV antigen that is conserved amoung SARSV isolates is preferred. In this regard, nonstructural protein (e.g., Pol, Hel, 3CLp, MP, PLP1, PLP2) may be particularly useful.

The present invention relates to kits for identifying individuals exposed to SARS and reagents used therein. The kits comprise a first container which contains antibodies which were produced in response to exposure to an antigen from SARS virus and a second container which contains the SARS antigen(s). The kits may be adapted for quantifying the amount of anti-SARS antibodies present in a sample of an individual. Such information may be used in determining the prognosis of an infected individual.

The present invention relates to methods of identifying individuals exposed to SARS virus, or biological samples containing SARSV, by detecting the presence of nucleic acid from SARS virus. Quantification of the amount of SARS nucleic acid present in a sample of an individual may be used in determining the prognosis of an infected individual. The methods utilize oligonucleotide probes and/or primers that are similar or complementary in sequence to the SARSV genome or transcription or replication products. Preferred probes and primers are described herein. Also included in the present invention are kits for carrying out the methods of detecting the SARSV nucleic acid.

The invention further includes a method for the treatment and/or prevention of SARS through the administration of a therapeutically effective amount of at least one antiviral compound from among those described in the US patents and published international patent applications listed in Table 1 and Table 2. In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In a further embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. In another embodiment, the antiviral compound is an inhibitor of an RNA-dependent RNA polymerase. In another embodiment, a first antiviral compound which is a protease inhibitor is administered with a second antiviral compound which is an RNA-dependent RNA polymerase inhibitor. The invention further provides for the administration of a steroidal anti-inflammatory drug in combination with at least one antiviral compound, for example, from the antiviral compounds described in the documents listed in Table 1 and Table 2.

The invention further provides for a method for the treatment and/or prevention of SARS through the administration of a therapeutically effective amount of at least one antiviral compound from among those described in the US patents and published international patent applications listed in Table 1 and Table 2 by inhalation. In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In a further embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. In another embodiment, the antiviral compound is an inhibitor of an RNA dependent RNA polymerase. In another embodiment, a first antiviral compound which is a protease inhibitor is administered with a second antiviral compound which is an RNA-dependent RNA polymerase inhibitor. The invention further provides for the administration of a steroidal anti-inflammatory drug in combination with at least one antiviral compound, for example, from the antiviral compounds described in the documents listed in Table 1 and Table 2 by inhalation. The steroidal anti-inflammatory drug may be administered by inhalation for a local effect or administered for systemic absorption such as via an oral or intravenous route.

The invention further provides the use of an antiviral compound, as defined above, in the manufacture of a medicament for the treatment of severe acute respiratory syndrome.

The invention further provides for a kit for use by a consumer for the treatment and/or prevention of SARS. Such a kit comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of at least one antiviral compound from among those described in the US patents and published international patent applications listed in Table 1 and Table 2 and a pharmaceutically acceptable carrier, vehicle or diluent; (b) a container for holding the pharmaceutical composition; and, optionally; (c) instructions describing a method of using the pharmaceutical compositions for the treatment and or the prevention of SARS. The kit may optionally contain a plurality of antiviral compounds for the treatment of SARS wherein the anti viral compounds are selected from 3C-like protease inhibitors and papain-like protease inhibitors. In a further embodiment, the kit contains an antiviral compound which is an RNA-dependent RNA polymerase inhibitor. When the kit comprises more than one antiviral compound, the antiviral compounds contained in the kit may be optionally combined in the same pharmaceutical composition.

An additional aspect of the invention provides for the use of at least one of the antiviral compounds described in the US patents and published international patent applications listed in Table 1 and Table 2 for the manufacture of a medicament for the treatment or prevention of SARS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic of coronavirus genome organization.

FIG. 2: Schematic of coronavirus ORF1a/ORF1b gene products.

FIG. 3(A-C): Alignment of coronavirus polynucleotide sequences for selected genes (including nucleocapsid (N), matrix (M), and hemagluttinin-esterase (HE)).

FIG. 4(A-F): Alignment of coronavirus polypeptide sequences (including ORF1a/ORF1b, nucleocapsid (NP), hemagluttinin-esterase (HE), envelope (Sm or E), matrix (M), and spike (S).

FIG. 5: Alignment of spike (S) polypeptide sequences, taken from FIG. 4, in the region of the junction of the S1 and the S2 domains, and protease cleavage site for selected coronaviruses.

FIG. 6: CDC phylogenetic tree of SARS-CoV strain (Clustalx 1.82, neighbor-joining tree).

FIG. 6A shows coronavirus N protein analysis,

FIG. 6B shows coronavirus S protein analysis, and

FIG. 6C shows coronavirus M protein analysis.

FIG. 7: Conserved and specific sequence of the SARS virus.

FIGS. 7A-7D show multiple sequence alignments (CLUSTAL W 1.82) of the structural proteins of the SARS virus genome (7A: PEP4 Spike protein; 7B: PEP7 small membrane protein; 7C: PEP8 matrix glycoprotein; 7D: PEP13 nucleocapsid protein), which have counterparts in all or some of the other known coronaviruses.

FIGS. 7E-7H show dendrograms reporting the protein distances among the sequences in alignments 7A-7D. Labels 229E: human coronavirus; MEV: murine hepatitis virus; TGV: transmissible gastroenteritis virus; AIBV: avian infectious bronchitis virus; BOVINE: Bovine coronavirus; PEDV: porcine epidemic diarrhea virus.

FIG. 8: Alignment of the 5′UTR of several coronaviruses, to show consensus nucleotide sequence at the 5′UTR.

FIG. 9: Sequences of preferred primers for amplification of the 5′UTR. F and R denote forward and reverse PCR primers, and the numbers indicate nucleotide positions withing FIG. 8.

FIG. 10: Alignment of the 3′UTR of several coronaviruses, to show consensus nucleotide sequence at the 3′UTR.

FIG. 11: Sequences of preferred primers for amplification of the 3′UTR. F and R denote forward and reverse PCR primers, and numbers indicate nucleotide positions within FIG. 10.

FIG. 12: Coiled-coil prediction for SEQ ID NO: 6042, using Coils program (FIG. 12A) or LearrCoil (FIG. 12B).

FIG. 13: Example of insertion of a reporter gene-of-interest at a site between exisiting SARS virus genes. Small nonstructural gene products are not depicted schematically.

FIG. 14: Schematic depicting representative examples of SARS virus replicons. Small nonstructural gene products are not depicted schematically.

FIG. 15: SARS virus nsp2 proteinase (3CLp) and identification of catalytic and substrate sites.

FIG. 16: alignment of SARS virus nsp2 proteinase (3CLp) with that of avian IBV, MHV, and BCoV. Residues in dotted boxes are key residues the substrate sites (F, Y & H); residues in solid boxes are catalytic cysteine (C) and histidine (H) residues.

FIG. 17: Genome organization of SARS coronavirus. Replicase and structural regions are shown, along with the predicted products of cleavage within ORF1a and ORF1b. The position of the 5′ RNA leader sequence (L), the 3′ poly(A) tract and the ribosomal frame-shift consensus between ORF1a and ORF1b are also indicated. Each box represent a protein product. They are shaded according to the level of amino acid identity with corresponding proteins of other coronaviruses (see also Table 2). The SARS-specific genes are white. Positions of the 9 SARS-specific six-base IG sequences (5′-ACGAAC-3′; SEQ ID NO 7293) are indicated by arrows.

FIG. 18: Genome organization of Coronaviruses representative of group 1 (HCoV-229E, accession number: AF304460), group 2 (mouse hepatitis virus MHV, accession number: NC_(—)001846), group 3 (avian infectious bronchitis virus AIBV, accession number: NC_(—)001451) and SARS coronavirus. Other completely sequenced coronaviruses used in this study are available at the following accession numbers: porcine epidemic diarrhea virus (PEDV), AF353511; transmissible gastroenteritis virus (TGV), ND_(—)002306; Bovine coronavirus (BCoV): AF220295. Red boxes represent group-specific genes. The position of the leader RNA sequence and poly(A) tract is also indicated in genomes where they are reported. The position of specific IG sequences is indicated by circles of different shades. In the SARS genome, we also find three IG sequences specific for group 2 coronavirus.

FIG. 19: Topological model predicted for the spike protein anchored to the viral membrane. Structural and predicted functional domains are indicated. The N-terminal region (S1) is predicted to contain the receptor binding domain. Two coiled coil regions within the S2 domain, partially superimposed to leucine zipper motifs are presumably involved in oligomerization. The hydrophobic domain is responsible for membrane anchoring.

FIG. 20: Phylogenetic tree obtained from the multiple sequence alignment of a 922 bp internal region of the pol gene from 12 coronaviruses and SARS. Numbers at the nodes represent the result of a bootstrap analysis and strongly support the branches. Sequences not available within the complete coronavirus genomes have been retrieved from GenBank at the following accession numbers: hemagglutinating encephalomyelitis virus of swine (PHEV), AF124988, Human OC43 virus (OC43), AF124989, canine coronavirus (CCV), AF124986, feline infectious peritonitis virus (FIPV), AF124987, turkey coronavirus (TCV), AF124991, syaloacryoadenitis virus of rats (SDAV), AF124990.

FIG. 21: 21A. Unrooted tree obtained from the alignment of consensus sequences of the group I and group II S1 domain of spike proteins (G1_cons and G2_cons) with those of a group 3 spike (AIBV) and the spike of SARS virus. The number indicates the result of a bootstrap analysis. The sequences used to generate the consensus profile from group 1 are: HcoV-229E, accession number P15423; porcine epidemic diarrhea virus (PEDV), acc no: NP_(—)598310; transmissible gastroenteritis virus (TGV), acc no: NP_(—)058424; Canine coronavirus (CCV), acc no: S41453; porcine respiratory virus (PRV), acc no: S24284; feline infectious peritonitis virus (FIPV), acc no: VGIH79. The sequences used to generate the consensus profile from group 2 are: mouse hepatitis virus (MHV), acc no: NP_(—)045300; Bovine coronavirus (BCoV), acc no: NP_(—)150077; Human coronavirus OC43, acc no: P36334; hemagglutinating encephalomyelitis virus of swine (PHEV), acc no: AAL80031; for group 3, only the sequence of the spike protein of avian infectious bronchitis virus (AIBV), acc no: AAO34396 was used. 21B: Schematic representation of cysteine positions in S1 domains of group 1, 2 and 3, compared to the SARS spike. Horizontal bars represent the S1 amino acid sequences (in the case of SARS and AIBV) or the consensus profiles (generated from group 1, G1 _cons, and from group 2, G2_cons). The length of the bars are not to scale. Relative cysteine positions are indicated by rectangle bars. Only cysteines perfectly conserved within each consensus are reported. Lines connect cysteines conserved between the SARS S1 domain and the consensus sequences as shown.

FIG. 22: illustration of a Neisseria Adhesin A protein (NadA).

FIG. 23: Raw translation from SARS coronavirus genome (reading frame +1).

FIG. 24: Raw translation from SARS coronavirus genome (reading frame +3)

FIG. 25: 1b and Spike open reading frames, separated by *.

FIG. 26: SARS growth in vero cells.

FIG. 27: chromatogram of the capture step of SARS coronavirus on Matrix Cellufine Sulfate Superformance 150/10. Analysis was on 100 ml coronoavirus harvest. The left Y axis shows absorbance at 280 nm. The right Y axis shows the gradient (% B). The X axis shows the volume (ml).

FIG. 28: Silver-stained MCS chromatography fractions. Lanes are: (1) marker; (2) coronavirus vero cell harvest; (3) coronavirus vero cell harvest, after 0.65 μm filtration; (4) flowthrough; (5) wash; (6) 20% peak (virus peak). Lanes were loaded with 1 μg of test protein.

FIG. 29: Western Blot of MCS chromatography fractions. Lanes are as described for FIG. 28.

FIG. 30: Linear density gradient ultracentrifugation, 15-60% sucrose (SW28, 2 hours, 20000 rpm). The graph shows protein concentration (▪) and sucrose concentration (♦).

FIG. 31: Silver-stained density gradient fractions on NuPage 4-12% Bis-Tris-Ge (Novex), reduced conditions, heated for 10 minutes at 70° C. Lanes are: (1) marker; (2) 20% peak MCS; (3) density gradient fraction 11; (4) density gradient fraction 12; (5) density gradient fraction 13; (6) density gradient fraction 14; (7) density gradient fraction 15; (8) density gradient fraction 16; (9) density gradient fraction 17. The bulk of proteins was in fractions 15 to 17. Lanes 2, 8 and 9 were loaded with 1 μg protein.

FIG. 32: Chromatogram of the Capture Step of SARS coronavirus on MCS. Details are as for FIG. 27, except that 200 ml harvest was used.

FIG. 33: Silverstain (left) and Western Blot (right) of chromatographic fractions. Lanes are as described for FIGS. 28 and 29, except that lane (6) is the 5% peak. Treatment before SDS-PAGE was at room temperature for 30 minutes.

FIG. 34: Density Gradient Ultracentrifugation, 15-40% sucrose (SW28, 2 hours, 20000 rpm). The graph shows protein concentration (▪) and sucrose concentration (♦).

FIG. 35: Silverstain (left) and Western Blot (right) of Density Gradient Ultracentrifugation fractions on NuPage 4-12% Bis-Tris-Ge (Novex), reduced conditions. Lanes are: (1) marker; (2) density gradient fraction 6; (3) density gradient fraction 7; (4) density gradient fraction 8; (5) density gradient fraction 9; (6) density gradient fraction 10; (7) density gradient fraction 15. Fractions 7-10 (lanes 3-6) contained pure coronavirus proteins. The bulk of impurities was in fraction 15 (lane 7). Lanes 2, 8 and 9 were loaded with ˜1 μg protein. Treatment before SDS-PAGE was at room temperature for 30 minutes.

FIG. 36: EM pictures of Density Gradient Fractions 8-10. FIG. 36A shows fraction 8; FIG. 36B shows fraction 9; FIG. 36C shows fraction 10.

FIG. 37: Spike/NadA fusion constructs.

FIGS. 38 and 39: Results of the expression in E. coli of S1_(L), S1_(L)-NadA and S1_(L)-NadA_(Δanchor).

FIG. 38 shows SDS-PAGE analysis of total lysates from BL21(DE3)/pET, BL21(DE3)/pET-S1_(L) and BL21 (DE3)/pET-S1_(L)-NadA_(Δanchor). The bands are indicated by an arrow, and the three lanes are, from left to right: BL21(DE3)/pET; BL21(DE3)/pET-S1_(L); BL21(DE3)/pET-S1_(L)-NadA_(Δanchor).

FIG. 39 shows (39A) SDS-PAGE and (39B) western blot analyses of total lysates from BL21(DE3)/pET, BL21(DE3)/pET-S1_(L)-NadA (grown under un-induced condition) and BL21(DE3)/pET-S1_(L)-NadA (grown under induced condition). The bands are indicated by an arrow, and lanes are, from left to right: BL21(DE3)/pET; BL21(DE3)/pET-S1_(L)-NadA; BL21(DE3)/pET-S1_(L)-NadA. The western blot shows the presence of oligomeric forms of the protein.

FIG. 40: Schematic of SARS Spike clones.

FIG. 41: Transient Expression of SARS Spike Proteins (western blot of COS7 cell lysate). Each lane of the 4-20% TG SDS gel was loaded with 20 μg cell lysate (total 1.2 mg). The labeling antibodies are shown.

FIG. 42: Western blot analyses of COS7 cell lysates on 4% TG SDS gel showing oligomerization state of intracellular S molecules.

FIG. 43: Western blot analyses of COS7 cell lysates on 4-20% TG SDS gel showing Transient Expression of SARS Spike Proteins. Lanes are: (1) mock, AF; (2) mock, DF; (3) nSh, AF; (4) nSh, DF; (5) nShΔTC, AF; (6) nShΔTC, DF. Each lane was loaded with 5 μl of each sample, 400 μl total. The blot was labeled with antibody against the His-tagged protein.

FIG. 44: Western blot analyses of COS7 cell medium on 4-20% TG SDS gel showing Transient Expression of SARS Spike Proteins. Truncated spike protein is secreted. Spike proteins were purified from the culture medium (from a 10 cm plate), first by a ConA column and then finally by His·tag Magnetic beads. Each lane was loaded with one third of the material.

FIG. 45: Western blot analyses of COS7 cell lysates on 4-20% TG SDS gel showing glycoslation of SARS spike proteins. In the two left-hand blots (lanes 1-5), samples were boiled in SDS and β-mercaptoethanol; in the two right-hand blots (lanes 6-11), samples were in SDS only, with no boiling. Lanes 1-8 were labeled with a monoclonal raised against the His-tag protein; labes 9-11 were labeled with rabbit anti-SARS antibody.

FIG. 46: Effect of SARS spike protein expression on cell viability.

FIG. 47: Western blot analyses of COS7 cell lysates on 4% TG SDS gels showing oligomerization state of intracellular spike molecules. Blots were labeled with anti-His-tag mAb. The membrane fraction of COS7 cell lysate was fractionated by a sizing column before loading the lanes. Fractions 7 to 14 show bands with kDa values of: 71000, 1400, 898, 572, 365, 232, 148 and 99, respectively.

FIG. 48: Fractionation of cells into aqueous and detergent fractions.

FIG. 49: Schematic of constructs for use in OMV preparation.

FIG. 50: SARS HR1 and HR2 constructs.

FIG. 51: Vaccine protection froms SARS in Balb/c mouse model.

FIG. 52: Expressed on Spike protein in transfected 293 cell lysates (52A) or COS7 cell culture supernatants (52B). Proteins were separated on 4-20% TG SDS gels. The label was anti-His-tag, except for the right-hand three lanes of 52B, where the label was rabbit anti-SARS serum.

In FIG. 52A, the left-hand three lanes were treated with DTT and were boiled, but neither treatment was used for the right-hand three lanes. In FIG. 52B, no DTT was used, but all lanes were heated to 80° C. for 5 minutes.

FIG. 53: Western blot of Spike proteins expressed in COS7 cells. Proteins were incubated at room temperature (RT), 80° C. or 100° C. to check for any effect on molecular weight. FIG. 54 shows similar experiments on SARS virions.

FIG. 55: Results of a pulse chase experiment, showing expression and processing of SARS spike protein following infection with alphavirus replicon particles. Cells were treated with or without EndoH as shown.

FIG. 56: Effect of heating on Spike protein trimers.

FIG. 57: Coomassie blue-stained gel of yeast-expressed proteins. Lanes are: 1-See Blue Standard (10 μl); 2-pAB24 gbl (20 μg); 3-SARS Spike S1 c.1 gbl (20 μg); 4-SARS Spike S1 c.2 gbl (20 μg); 5-See Blue Standard (10 μl ); 6-pAB24 ip (5 μl ); 7-SARS Spike S1 c.1 (5 μl ); 8-SARS Spike S1 c.2 (5 μl).

FIGS. 58 to 64: Schematics of preparation of yeast expression constructs.

FIGS. 65 to 66: Yeast-expressed sequences for Spike.

FIG. 67: Western blots showing expression of SARS spike protein from alphavirus replicon particles and replicon RNA.

FIG. 67A was run under non-reducing conditions and at room temperature (i.e. no heating), with lanes: (1) VEE/SIN-spike infection; (2) VEE/SIN-GFP infection; (3) Replicon-spike RNA transfection; (4) Replicon-GFP RNA transfection.

FIG. 67B was run with SARS virions at different temperatures, as shown.

FIG. 68: induction of antibody responses in mice. Vaccine groups are: (1) Inactivated SARS Virus; (2) Truncated Recombinant Spike Protein; (3) Full length Spike: DNA+DNA.PLG+ Alphavirus; (4) Full length Spike: Alphavirus particles only.

FIG. 69: Binding of human monoclonal antibody S3.2 to purified truncated Spike protein. The X-axis shows antibody concentration, and the Y-axis shows ELISA absorbance. The interpolation result is 2158.13.

FIG. 70: Geometric mean ELISA titers of antibodies induced by the SARS-CoV spike protein delivered as different vaccines (left to right: inactivated virus; 3 μg truncated spike protein; 75 μg DNA encoding truncated spike protein.

FIG. 71: Neutralization titers after immunization with (left) nSdΔTC protein or (right) DNA encoding nSdΔTC, delivered on PLG.

FIG. 72: Correlation between the spike antigen binding and neutralizing antibodies

FIG. 73: Western blot of CHO cell lines expressing Spike protein in full-length form (left) or in truncated form (right). Proteins were separated by 4-12% SDS-PAGE, with boiling in DTT and staining by polyclonal serum.

FIG. 74: Structural components of SARS-CoV spike glycoprotein and expression construct. L denotes leader peptide (residues 1-13), TM the transmembrane, and Cy the cytoplasmic tail segments. The hexa-His tags are not shown.

FIG. 75: Western blot analysis of SARS spike proteins expressed in COS7 cells.

In FIG. 75A, COS7 cells were transfected with indicated plasmid constructs and the expressed proteins in cell lysates 48 hr post-transfection were analysed by SDS-PAGE (4-20% polyacrylamide) in reducing and denaturing condtions, with proteins visualized by anti-histidine Mab.

In FIG. 75B, proteins were collected from cell culture medium 48 hr post-transfection and purified first by a ConA column and then by His-tag magnetic beads. Purifed proteins were analysed by SDS-PAGE (4-20% polyacrylamide) and were visualized by anti-SARS rabbit serum.

FIG. 76: Endo H sensitivity of C-terminal truncated spike protein (SΔ) found in cell lysate (lanes 1,2) and culture medium (lanes 3,4). Positions of internal SΔ protein and secreted SΔ protein are marked with arrow heads.

FIG. 77: Oligomeric status of the SARS spike protein. Recombinant S protein oligomer in COS7 cells transfected with the full-length spike construct (nSh). The cell lysates were treated with DTT and/or heat as indicated above each lane. The different forms of S protein in treated and untreated samples were visualized by SDS-PAGE (4% polyacrylamide) and Western blot analysis using anti-histidine MAb.

FIG. 78: Effect of heat denaturation on the oligomeric status of recombinant S protein in the absence of DTT. The COS7 cell lysates were heated before the electrophoresis as indicated and the S proteins were visualized as described fogi FIG. 77.

FIG. 79: Effect of heat denaturation on the oligomeric status of spike protein in SARS virion particles. SARS-CoV were grown in Vero cells, purified and solubilized from the virion particles by SDS, heat-denatured as indicated and visualized as described in FIG. 77, except that rabbit antiserum against the purified virus was used as a probe.

FIG. 80: Analysis of the oligomeric status of SARS virion spike protein by cross-linking experiment. Solubilized SARS virion proteins were treated with DMS. Both untreated (−) and DMS treated (+) virion proteins were heat denatured in the absence of DTT and visualized by 4% PAGE followed by silver staining.

FIGS. 81 & 82: Analysis of the oligomeric status of truncated spike protein by heat denaturation. Truncated spike protein within COS7 cell lysates (81) or secreted into culture medium (82) were heat denatured as indicated in the absence of DTT and visualized by Western blot analysis.

FIG. 83: Reactivity of deglycosylated full-length spike oligomer with conformational and non-conformational antibody. The full-length recombinant spike oligomer was partially deglycosylated with PNGase F in non denaturating condition and visualized by Western blot analysis using anti-histidine Mab (lane 1,2,3) or rabbit antiserum against purified SARS CoV (lane 4,5,6).

FIG. 84: Localization of expressed SARS spike proteins in fractionated COS7 cell lysate visualized by western blot. Cells were transfected with indicated plasmids and lysed with Dounce homogeniser in hypotonic buffer 48 hr post transfection. Cell lysate was centrifuged to obtain soluble cytosol and insoluble membrane fraction that was further solublized by 4% Triton X-100. Proteins were heated with SDS at 80 C and analysed by SDS-PAGE (4-20% polyacrylamide) in reducing condtion. Proteins were visualized by anti-histidine Mab. The cytosol fractions were loaded in lanes 1, 3, and 5 and the membrane fractions were loaded in lanes 2, 4, and 6.

FIG. 85: Intracellular and surface expression of recombinant full-length (A,D) or truncated (B,E) spike protein in COS7 cells. The cells were fixed at 48 hrs posttransfection and either treated with detergent (Cytofix/perm, BD Biosciences) for intracellular immunofluorescence (A,B,C) or with 2% paraformaldehyde for cell surface immunofluorescence observation (D,E,F) at ×40 magnification. Mock transfected cells (C,F) were included as controls.

FIGS. 86-105: SDS-PAGE od E. coli expressed proteins. Tot=total protein; Sol=soluble protein fraction. Labels are protein names (Tables 26-30).

FIG. 106: Immunofluorescence after administration of vector encoding optimsed N antigen.

FIG. 107: Immunofluorescence of (A) native and (B) codon-optimsed M sequences.

FIG. 108: Immunofluorescence of (A) native and (B) codon optimsed E sequences.

FIGS. 109-111: Western blots of Vero cells using rabbit antibodies obtained after immunization with spike proteins expressed in E. coli.

FIG. 112: Spike protein expression in 293 cells. Lanes: (M) Markers; (1) Mock transfected; (2,6) cells expressing nS protein, lysate; (3,7) cells expressing nSdTC protein, lysate; (4,8) cells expressing nS protein, supernatant; (5,9) (4) cells expressing nSdTC protein, supernatant. Staining antibody: (2 to 5) mouse serum obtained after DNA immunization; (6 to 9) rabbit serum obtained after immunization with whole killed virus.

FIG. 113: Six reading frames of SEQ ID NO: 9968.

FIG. 114: Six reading frames of SEQ ID NO: 10033.

FIG. 115: Alignment of bovine coronavirus pol 1ab (top row; SEQ ID NO: 10068), avian infectious bronchitis pol 1ab (second row; SEQ ID NO: 10069), murine hepatitis virus pol 1ab (third row; SEQ ID NO: 10070), SEQ ID NO^(S): 9997/9998 (fourth row) and a consensus sequence (bottom row; SEQ ID NO: 10071).

FIG. 116: Schematic of coronavirus genome organization.

FIG. 117: Schematic of coronavirus ORF1a/ORF1b gene products, including “*” region.

FIG. 118: Alignment.

FIG. 119: Alternative start codons within SEQ ID NO: 10080.

FIG. 120: Six reading frames of SEQ ID NO: 10084.

FIG. 121: Alignment of SEQ ID NO: 10033 and SEQ ID NO: 10084.

FIG. 122: Reading frames in SEQ ID NO: 10084.

FIG. 123: Start codon analysis for SEQ ID NO: 10084.

FIG. 124: BLAST analysis of SEQ ID NO: 10210.

FIG. 125: Epitope analysis of SEQ ID NO: 10210 by either (13A) Hopp & Woods or (13B) Kyte & Doolittle.

FIG. 126: Reading frames in SEQ ID NO: 10299.

FIG. 127: Reading frames in SEQ ID NO: 10505.

FIG. 128: Reading frames in SEQ ID NO: 11563.

FIG. 129: Reading frames in SEQ ID NO: 10033.

FIG. 130: Alignment of SEQ ID NO: 9997 and SEQ ID NO: 10033.

FIG. 131: Reading frames in SEQ ID NO: 10299.

FIG. 132: Reading frames in SEQ ID NO: 10505.

FIG. 133: Western Blot of SARS protease purification fractions.

FIG. 134: Cleavage of DABCYL-EDANS (a fluorescent tagged peptide with a SARS protease cleavage site) by SARS protease at different concentrations. The graph shows activity/concentration correlations with no protease (♦), 0.95 uM protease (▪) and 2.86 uM protease (●).

In the event of a discrepancy between a sequence in the sequence listing and a sequence in the drawings, the drawings should take precedence.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed., CRC Press, 1997); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag); Peters and Dalrymple, Fields Virology (2d ed), Fields et al. (eds.), B.N. Raven Press, New York, N.Y.

All publications, patents and patent applications cited herein, are hereby incorporated by reference in their entireties.

Severe Acute Respiratory Syndrome (SARS) virus has recently been identified as a new viral species. The SARS viral species includes the following isolates.

-   -   two virus isolates described in Peiris et al. “Coronavirus as a         possible cause of severe acute respiratory syndrome” Lancet         published online at         http://image.thelancet.com/extras/03art3477web.pdf on Apr. 8         2003, incorporated herein by reference in its entirety and the         sequences deposited with GenBank at accession number AY268070.     -   the isolates and viral sequences described in Drosten et al.,         “Identification of a Novel Coronavirus in Patients with Severe         Acute Respiratory Syndrome”, New England Journal of Medicine,         published online at http://www.nejm.org on Apr. 10, 2003.     -   the isolates and viral sequences described on the website of the         WHO network on Mar. 25 and 24, 2003.     -   the isolates and viral sequences described in Tsang et al., “A         Cluster of Cases of Severe Acute Respiratory Syndrome in Hong         Kong” New England Journal of Medicine, published online at         http://www.nejm.org on Mar. 31, 2003.     -   the isolates and viral sequences described in Poutanen et al.,         “Identification of Severe Acute Respiratory Syndrome in Canada”         New England Journal of Medicine, published online at         http://www.nejm.org on Mar. 31, 2003.         As described in the Lancet article, a 646 base pair         polynucleotide from the SARS virus has weak homology to viruses         of the family Cornoaviridae. The Lancet article further reports         that a deduced amino acid sequence (of 215 amino acids) from         this sequence has about 57% sequence homology to the RNA         polymerase of bovine coronavirus and murine hepatitis virus.         Phylogenetic analysis of the protein sequences are also         presented in the Lancet article showing that the polymerase         sequence is most closely related to the group II coronaviruses.

Additional SARS viral isolates can be identified, isolated and/or sequenced by virologists skilled in the art. Virologists can readily identify new viral isolates as a SARS virus. Criteria which a virologist may use to identify new SARS isolates include: sequence homology of the new isolate to known SARS viral isolates; similar genomic organization of the new viral isolate to known SARS viral isolates; immunological (serologic) similarity or identity with known SARS viral isolates; pathology; and similarity of virion morphology with known SARS viral isolates; and similarity of infected cell morphology as that caused by known SARS viral isolates (visualized, for instance, by electron microscopy).

Methods for isolating and sequencing SARS viral isolates include the methods described by Peiris et al. in the Lancet paper. As reported in the Lancet paper, RNA from clinical samples can be reverse transcribed with random hexamers and cDNA can be amplified with primers having sequences of SEQ ID NOS: 6584 & 6585 in the presence of 2.5 mmol/L magnesium chloride (94° C. for 1 min, 50° C. for 1 min, and 72° C. for 1 min).

Reverse transcription of a viral isolate using random hexamers can be accomplished in an RT-PCR assay as follows. Virus isolates are propagated on mammalian cells, particularly fetal rhesus kidney cells. Total RNA from virus-infected and virus-uninfected fetal rhesus kidney cells is then isolated. RNA samples are reverse transcribed with a primer having SEQ ID NO: 6586. cDNA can be amplified by a primer having SEQ ID NO: 6587. Unique PCR products (in size) in the infected cell preparation are then cloned and sequenced, and genetic homology of the sequence compared with those in GenBank.

One skilled in the art would be able to identify and clone additional genomic regions using a variety of standard cloning techniques, such as, for example, using random primer RT-PCR and detection of sequences overlapping one or more of the above sequences, and/or using oligonucleotide primers, e.g., degenerate primers, based on the sequences provided herein (see FIGS. 1-5, FIGS. 8-11, SEQ ID NOS: 3-20).

Cloning, sequencing and identification of SARS virus by one skilled in the art can be further facilitated by the use of polynucleotide sequences, particularly RNA polymerase sequences, from related Coronaviruses.

Sequence homology of new viral isolates with the known SARS isolates described above can be readily determined by one skilled in the art. New SARS isolates may be identified by a percent homology of viral nucleotide sequences of 99%, 95%, 92%, 90%, 85%, or 80% homology of the new virus to known SARS viral polynucleotide sequences. New SARS isolates may also be identified by percent homology of 99%, 95%, 92%, 90%, 85%, or 80% homology of the polypeptides encoded by the polynucleotides of the new virus and the polypeptides encoded by known SARS virus.

New SARS isolates may also be identified by a percent homology of 99%, 95%, 92%, 90%, 85%, or 80% homology of the polynucleotide sequence for specific genomic regions for the new virus with the polynucleotide sequence for specific genomic regions of the known SARS viruses. Additionally, new SARS isolates may be identified by a percent homology of 99%, 95%, 92%, 90%, 85%, or 80% homology of the polypeptide sequence encoded by the polynucleotide of specific genomic regions of the new SARS virus to the polypeptide sequence encoded by the polynucleotides of specific regions of the known SARS virus. These genomic regions may include regions (e.g., gene products) which are typically in common among numerous coronaviruses, as well as group specific regions (e.g., antigenic groups), such as, for example, any one of the following genomic regions which could be readily identified by a virologist skilled in the art: 5′untranslated region (UTR), leader sequence, ORF1a, ORF1b, nonstructural protein 2 (NS2), hemagglutinin-esterase glycoprotein (HE) (also referred to as E3), spike glycoprotein (S) (also referred to as E2), ORF3a, ORF3b, ORF3x, nonstructural protein 4 (NS4), envelope (small membrane) protein (E) (also referred to as sM), membrane glycoprotein (M) (also referred to as E1), ORF5a, ORF5b, nucleocapsid phosphoprotein (N), ORF7a, ORF7b, intergenic sequences, 3′UTR, or RNA dependent RNA polymerase (pol). The SARS virus may have identifiable genomic regions with one or more the above-identified genomic regions. A SARS viral antigen includes a protein encoded by any one of these genomic regions. A SARS viral antigen may be a protein or a fragment thereof, which is highly conserved with coronaviruses. A SARS viral antigen may be a protein or fragment thereof, which is specific to the SARS virus (as compared to known cornaviruses). (See, FIGS. 1-5, FIGS. 8-11, SEQ ID NOS: 3-20).

One skilled in the art could also recognize electron microscopy of a SARS virus infected mammalian cell. Electron microscopy of SARS infected cells are shown in the Lancet paper. As discussed in the paper, electron microscopy of negative stained (3% potassium phospho-tungstate, pH 7.0) ultracentrifuged cell-culture extracts of SARS infected fetal rhesus kidney cells show the presence of pleomorphic enveloped virus particles of around 80-90 nm (range 70-130 nm) in diameter with surface morphology compatible with a coronavirus (see Lancet paper, FIG. 1). Thin-section electron microscopy of infected cells reveals virus particles of 55-90 nm diameter within smooth walled vesicles in the cytoplasm (see Lancet paper, FIG. 2B). Electron microscopy can also be used to observe virus particles at the cell surface. Electron microscopy of a human lung biopsy sample depicts similar viral morphology. See Lancet paper FIG. 2A.

I. SARS Polypeptides and Polynucleotides

The invention relates to nucleic acids and proteins from SARS virus. Such polynucleotides and polypeptides are exemplified further below.

In one embodiment, the polynucleotides of the invention do not include one of the following five primers, disclosed at http://content.nejm.org/cgi/reprint/NEJMoa030781v2.pdf: SEQ ID NOs: 6034-38.

The invention also includes polynucleotide sequences which can be used as probes for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified in SEQ ID NOS: 21-1020. The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in SEQ ID NOS: 21-1020.

The invention includes a polypeptide sequence comprising an amino acid sequence from the sequence shown in FIG. 23. Such amino acid sequences are SEQ ID NOS: 6588-6809. The invention includes polypeptides comprising an amino acid sequence having sequence identity to these sequences, and the invention includes a fragment of a polypeptide comprising one of these sequences.

The invention includes a polypeptide comprising an amino acid sequence from the sequence shown in FIG. 24. Such amino acid sequences are SEQ ID NOS: 6810-7179. The invention includes a protein comprising an amino acid sequence having sequence identity to these sequences, and the invention includes a fragment of a protein comprising one of these sequences.

The invention includes a protein comprising SEQ ID NO: 6039. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 6039. The invention includes a fragment of a polypeptide comprising SEQ ID NO: 6039. The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 6039, or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding SEQ ID NO: 6039, or a fragment thereof. The invention includes an immunogenic composition comprising a polypeptide comprising SEQ ID NO: 6039, or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 6039, or a fragment thereof. SEQ ID NO: 6039 demonstrates functional homology with ORF1a of coronaviruses.

Predicted transmembrane or hydrophobic regions of SEQ ID NO: 6039 are identified below. Although the polyprotein of coronaviruses is proteolytically cleaved into numerous smaller proteins, hydrophobic domains in the polyprotein are known to mediate the membrane association of the replication complex and to be able to dramatically alter the architecture of host cell membranes. Accordingly, the hydrophobic domains of the polyprotein are targets for genetic mutation to develop attenuated SARS virus vaccines. The hydrophobic domains are also targets for small molecule inhibitors of the SARS virus. The hydrophobic domains may also be used to generate antibodies specific to those regions to treat or prevent SARS virus infection.

Predicted Transmembrane Helices in SEQ ID NO: 6039

The sequence positions in brackets denominate the core region.

Only scores above 500 are considered significant. from to score center Inside to outside helices: 43 found 100 (100) 118 (116) 103 107 473 (473) 488 (488) 1003 481 529 (532) 549 (549) 541 539 584 (584) 606 (601) 1049 594 773 (773) 791 (789) 514 782 1071 (1071) 1089 (1086) 243 1078 1121 (1121) 1137 (1137) 459 1130 1679 (1679) 1696 (1696) 404 1686 2098 (2102) 2119 (2116) 509 2109 2145 (2145) 2160 (2160) 797 2153 2206 (2209) 2224 (2224) 2686 2216 2316 (2316) 2332 (2332) 2123 2325 2335 (2339) 2358 (2354) 2101 2346 2373 (2373) 2390 (2390) 532 2380 2597 (2600) 2615 (2615) 307 2607 2753 (2753) 2770 (2768) 2242 2760 2831 (2833) 2854 (2851) 759 2841 2879 (2882) 2900 (2897) 526 2889 2990 (2996) 3012 (3010) 1289 3003 3024 (3024) 3042 (3039) 2281 3032 3054 (3058) 3075 (3072) 2536 3065 3105 (3109) 3127 (3123) 2010 3116 3143 (3143) 3163 (3159) 184 3152 3253 (3255) 3272 (3272) 319 3262 3346 (3346) 3366 (3366) 203 3356 3375 (3375) 3392 (3392) 305 3384 3438 (3438) 3455 (3453) 1021 3445 3559 (3567) 3584 (3581) 1885 3574 3589 (3589) 3606 (3604) 2018 3596 3611 (3611) 3629 (3629) 2304 3621 3659 (3659) 3674 (3674) 1561 3667 3756 (3758) 3777 (3774) 2352 3767 3890 (3890) 3904 (3904) 485 3897 3916 (3919) 3934 (3934) 241 3926 4035 (4035) 4051 (4051) 335 4044 4217 (4217) 4232 (4232) 272 4224 4239 (4239) 4257 (4254) 402 4247 Outside to inside helices: 43 found 94 (97) 118 (112) 291 104 400 (400) 418 (415) 243 407 473 (473) 488 (488) 1113 481 523 (528) 548 (548) 285 538 583 (583) 606 (601) 662 593 776 (776) 791 (791) 1435 783 1068 (1071) 1089 (1086) 370 1078 1121 (1121) 1137 (1137) 455 1130 1679 (1679) 1696 (1694) 340 1686 2098 (2098) 2119 (2116) 678 2109 2148 (2148) 2163 (2163) 434 2155 2208 (2210) 2231 (2226) 2389 2219 2309 (2309) 2332 (2326) 1773 2318 2342 (2342) 2368 (2360) 1666 2353 2373 (2373) 2390 (2390) 254 2380 2753 (2755) 2770 (2770) 2119 2763 2832 (2835) 2854 (2851) 687 2844 2858 (2858) 2873 (2873) 253 2866 2879 (2882) 2899 (2899) 400 2889 2990 (2990) 3005 (3005) 875 2998 3020 (3024) 3042 (3042) 2795 3032 3059 (3059) 3075 (3075) 2137 3067 3105 (3108) 3127 (3123) 1902 3115 3142 (3145) 3162 (3162) 540 3152 3343 (3351) 3366 (3366) 496 3358 3437 (3437) 3453 (3453) 848 3444 3489 (3491) 3508 (3505) 302 3498 3560 (3560) 3577 (3577) 1460 3569 3591 (3591) 3606 (3606) 2193 3598 3610 (3610) 3627 (3627) 1484 3620 3656 (3658) 3678 (3675) 1240 3668 3681 (3684) 3701 (3699) 590 3691 3710 (3713) 3738 (3728) 1696 3721 3723 (3723) 3738 (3738) 1670 3730 3760 (3760) 3777 (3775) 2367 3767 3881 (3884) 3902 (3900) 249 3892 4099 (4099) 4114 (4114) 389 4106 4234 (4234) 4254 (4249) 325 4241 4338 (4341) 4360 (4360) 505 4348

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6039, wherein said fragment comprises an amino acid sequence including one or more of the hydrophobic transmembrane sequences identified above. The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6039 wherein said fragment comprises one or more of the following polypeptide sequences of SEQ ID NO: 6039: 473-488; 529-549, 584-606, 773-791, 2098-2119, 2145-2160, 2206-2224, 2316-2332, 2335-2358, 2373-2390, 2753-2770, 2831-2854, 2879-2990, 2990-3012, 3024-3042, 3054-3075, 3105-3127, 3438-3455, 3559-3584, 3589-3606, 3611-3629, 3659-3674, 3756-3777, 473-488, 583-606, 776-791, 2098-2119, 2208-2231, 2309-2332, 2342-2368, 2753-2770, 2832-2854, 2990-3005, 3020-3042, 3059-3075, 3105-3127, 3142-3162, 3437-3453, 3560-3577, 3591-3606, 3610-3627, 3656-3678, 3710-3738, 3723-3738, and 3760-3777. Preferably, the fragment comprises one or more of the following polypeptide sequences of SEQ ID NO: 6039: 2206-2224, 2316-2332, 2335-2358, 2753-2770, 3024-3042, 3054-3075, 3105-3127, 3589-3606, 3611-3629, 3756-3777, 2208-2231, 2753-2770, 3020-3042, 3059-3075, and 3591-3606. Preferably, the fragment comprises one or more of the following polypeptide sequences of SEQ ID NO: 6039: 2206-2224 and 3020-3042. The invention also includes polynucleotides encoding each of the polypeptide fragments identified above.

The invention includes an attenuated SARS virus wherein said attenuated SARS virus contains an addition, deletion or substitution in the polynucleotides encoding for one of the hydrophobic domains identified above. The invention also includes a method for creating an attenuated SARS virus comprising mutating a SARS virus by adding, deleting or substituting the viral genome of the SARS virus to alter the coding of one or more of the hydrophobic domains of SEQ ID NO: 6039 identified above.

The invention includes an antibody which specifically identifies one or more of the hydrophobic regions of SEQ ID NO: 6039 identified above. The invention includes a small molecule which binds to, interferes with the hydrophobicity of or otherwise disrupts one or more of the hydrophobic regions of SEQ ID NO: 6039 identified above.

Predicted N-glycosylation sites of SEQ ID NO: 6039 are identified in the chart below.

Prediction of N-glycosylation Sites in SEQ ID NO: 6039 Jury NGlyc Position Potential agreement result 48 NGTC SEQ ID NO: 7180 0.6371 (7/9) + 389 NHSN SEQ ID NO: 7181 0.6132 (6/9) + 916 NFSS SEQ ID NO: 7182 0.5807 (7/9) + 1628 NHTK SEQ ID NO: 7183 0.5610 (7/9) + 1696 NKTV SEQ ID NO: 7184 0.5297 (5/9) + 2031 NPTI SEQ ID NO: 9764 0.5299 (5/9) + WARNING: PRO- X1. 2249 NSSN SEQ ID NO: 7185 0.6329 (9/9) ++ 2459 NPTD SEQ ID NO: 9765 0.5599 (6/9) + WARNING: PRO- X1. 2685 NVSL SEQ ID NO: 7186 0.6071 (8/9) + 4233 NATE SEQ ID NO: 7187 0.6144 (7/9) +

Accordingly, the invention comprises a fragment of SEQ ID NO: 6039 wherein said fragment comprises an amino acid sequence which includes one or more of the N-glycosylation sites identified above. Preferably, the fragment comprises one or more sequences selected from the group consisting of SEQ ID NOS: 7180-7187 & 9764-9765. Preferably, the fragment comprises the amino acid sequence NSSN (SEQ ID NO: 7185).

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6039 wherein said fragment does not include one or more of the glycosylation sites identified above. The invention also includes a polynucleotide encoding such a polypeptide.

T-epitopes for SEQ ID NO: 6039 are identified in Table 13. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified as SEQ ID NOS: 7400-7639; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified as SEQ ID NOS: 7400-7639, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus.

The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The ORF1a and ORF1b sequences of coronaviruses are typically translated as a single ORF1ab polyprotein. Slippage of the ribosome during translation generates an a-1 frameshift. One region of such slippage is illustrated below:    gggttttacacttagaaacacagtctgtaccgtctgcggaatgtggaaaggttatggctgtagttgtga +1   G  F  T  L  R  N  T  V  C  T  V  C  G  M  W  K  G  Y  G  C  S  C  D +3  G  F  Y  T  -  K  H  S  L  Y  R  L  R  N  V  E  R  L  W  L  -  L  -    ccaactccgcgaacccttgatgcagtctgcggatgcatcaacgtttttaaacgggtttgcggtgtaagt +1   Q  L  R  E  P  L  M  Q  S  A  D  A  S  T  F  L  N  G  F  A  V  -  V +3  P  T  P  R  T  L  D  A  V  C  G  C  I  N  V  F  K  R  V  C  G  V  S    gcagcccgtcttacaccgtgcggcacaggcactagtactg   (SEQ ID NO: 7224) +1   Q  P  V  L  H  R  A  A  Q  A  L  V  L    (SEQ ID NOS: 7225-7226) +3  A  A  R  L  T  P  C  G  T  G  T  S  T     (SEQ ID NOS: 7227-7229)

which would generate the following translational slippage (SEQ ID NOS: 7230-7231): ccaactccgcgaacccttgatgcagtctgcggatgcatcaacgtttttaaacgggtttgcggtgtaagt  Q  L  R  E  P  L  M  Q  S  A  D  A  S  T  F  L  N  R  V  C  G  V  S

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 7232. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 7232. The invention includes a fragment of a polypeptide comprising SEQ ID NO: 7232 The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 7232 or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding SEQ ID NO: 7232 or a fragment thereof. The invention includes an immunogenic composition comprising a polypeptide comprising SEQ ID NO: 7232 or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 7232 or a fragment thereof.

The invention also includes a polypeptide comprising amino acid sequence X₁—X₂—X₃, where X₁ is SEQ ID NO: 7233, X₂ is from one to ten amino acids, and X₃ is SEQ ID NO: 7234. X₂ can comprise any sequence of one to ten amino acids (SEQ ID NOS: 7235-7244) but, in preferred embodiments, X₂ is selected from the group consisting of F, FL, FLN, FLNR (SEQ ID NO: 7245), FLNRV (SEQ ID NO: 7246) and FLNRVC (SEQ ID NO: 7247). Preferably, X₂ is SEQ ID NO: 7247. These preferred embodiments are shown as SEQ ID NOS: 7248-7253.

The invention includes a polypeptide comprising an amino acid sequence having sequence identity to said amino acid sequences X₁—X₂—X₃. The invention includes a fragment of a polypeptide comprising said amino acid sequences X₁—X₂—X₃. The invention includes a diagnostic kit comprising a polypeptide comprising said amino acid sequences X₁—X₂—X₃ or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding said amino acid sequences X₁—X₂—X₃ or a fragment thereof. The invention includes an immunogenic composition comprising a polypeptide comprising said amino acid sequences X₁—X₂—X₃ or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising said amino acid sequences X₁—X₂—X₃ or a fragment thereof.

The amino acid sequences X₁—X₂—X₃ (i.e. SEQ ID NOS: 7235-7244) demonstrate functional homology with the polyprotein of murine hepatitis virus. This polyprotein is cleaved to produce multiple proteins. Proteins which can be generated from the X₁—X₂—X₃ polyprotein, where X₂ is six amino acids (SEQ ID NO: 7240) are listed below. Coordinates in Mouse virus protein Coordinates in Mouse virus SEQ ID NO: 7240 Nsp2 3334-3636 3241-3546 Nsp3 3637-3923 3547-3836 Nsp4 3924-4015 (or 4012) 3837-3919 Nsp5 4016 (or 4013)-4209 3920-4117 Nsp6 4210-4319 4118-4230 Nsp7 4320-4456 4231-4369 Nsp9 4457-5384 4370-5301 Nsp10 5385-5984 5302-5902 Nsp11 5985-6505 5903-6429 Nsp12 6506-6879 6430-6775 Nsp13 6880-7178 6776-7073

The invention includes a fragment of the amino acid sequence X₁—X₂—X₃ (i.e. SEQ ID NOS: 7235-7244) wherein the fragment comprises one of the polypeptide sequences identified in the above table. The invention further includes a fragment of the amino acid sequence X₁—X₂—X₃ wherein said fragment comprises a polypeptide sequence which has a serine at its N-terminus and a glutamine at its C-terminus. The invention further includes a fragment of the amino acid sequence X₁—X₂—X₃ wherein said fragment comprises a polypeptide sequence which has an Alanine at its N-terminus and a glutamine at its C-terminus. The invention further includes a fragment of the amino acid sequence X₁—X₂—X₃ wherein said fragment comprises a polypeptide sequence which has a Asparagine at its N-terminus and a glutamine at its C-terminus. The invention further includes a fragment of the amino acid sequence X₁—X₂—X₃ wherein said fragment comprises a Cysteine at its N-terminus and a Glutamine at its C-terminus. Each of the fragments identified above can be used in fusion proteins.

The invention includes a diagnostic kit comprising a polypeptide comprising at least one of the fragments of the amino acid sequence X₁—X₂—X₃ (i.e. SEQ ID NOS: 7235-7244) identified in the above paragraph. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding at least one of the fragments of the amino acid sequence X₁—X₂—X₃ identified in the above paragraph. The invention includes an immunogenic composition comprising a polypeptide comprising at least one of the fragments of the amino acid sequence X₁—X₂—X₃ identified in the above paragraph. The invention includes an antibody which recognizes a polypeptide comprising at least one of the fragments of the amino acid sequence X₁—X₂—X₃ identified in the above paragraph.

Predicted N-glycosylation sites of the amino acid sequence X₁—X₂—X₃ when X₂ is six amino acids are identified at the asparagines located at the following amino acid positions 48; 389; 556; 916; 1628; 1696; 1899; 2079; 2249; 2252; 2507; 2685; 3303; 3373; 3382; 3720; 4150; 4233; 4240; 5016; 5280; 5403; 5558; 5650; 5905; 6031; 6130; 6474; 6918; 6973. Accordingly, the invention comprises a fragment of SEQ ID NO: 7239 wherein said fragment is at least ten amino acids and wherein said fragment comprises one or more of the asparagines from the amino acid positions of SEQ ID NO: 7239 selected from the group consisting of 8; 389; 556; 916; 1628; 1696; 1899; 2079; 2249; 2252; 2507; 2685; 3303; 3373; 3382; 3720; 4150; 4233; 4240; 5016; 5280; 5403; 5558; 5650; 5905; 6031; 6130; 6474; 6918; and 6973.

A zinc binding region 2 site within SEQ ID NOS: 7235-7244 is identified at amino acid residues 2102-2112 (SEQ ID NO: 7254 HGIAAINSVPW). The polypeptide of SEQ ID NOS: 7235-7244 will be processed by the SARS virus into multiple peptides. This zinc binding region falls within the nsp1 region of the polypeptide. SEQ ID NO: 7254 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprises SEQ ID NO: 7254. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 7254. The invention includes a method of screening SEQ ID NO: 7254 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 7254 in a host cell. The invention includes a fragment of SEQ ID NOS: 7235-7244, wherein said fragment comprises SEQ ID NO: 7254. The invention includes a polypeptide comprising SEQ ID NO: 7254 wherein said polypeptide is complexed with a zinc ion. The invention includes a small molecule which prevents a zinc ion from complexing with the polypeptide of SEQ ID NO: 7254. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 7254.

The polyprotein encoded by the SARS virus will contain at least two protease domains: a papain-like cystein protease (PLP) and a chymotrypsin-picornavirus 3C-like protease (3CLp). (There may be more than one copy of the PLP domain). These proteases function to cleave the polyprotein into multiple smaller proteins. The 3C-like protease, also known as the “main protease” or Mpro, is itself cleaved from the polyprotein by its own autoprotease activity. See generally, Chapter 35 of Fields Virology (2nd ed), Fields et al. (eds.), B.N. Raven Press, New York, N.Y., and Anand et al., EMBO Journal (2002) 21 (13): 3213-3224. This 3CLp generally corresponds with the Nsp2 region identified above.

The SARS virus 3CLp protein is further characterized by SEQ ID NO: 6569 (also SEQ ID NO: 9769), as shown in FIG. 15.

FIG. 16 also illustrates the SARS virus 3CLp, in allignment with the 3CLp of avian infectious bronchitis (IBV; SEQ ID NO: 6570), mouse hepatitis virus (MHV; SEQ ID NO: 6571), and bovine coronavirus (BCoV; SEQ ID NO: 6572). Accordingly, the invention includes a polypeptide sequence comprising SEQ ID NO: 6569, or a fragment thereof, or a polypeptide sequence having sequence identity thereto. The invention further includes a polynucleotide sequence encoding SEQ ID NO: 6569, or a fragment thereof. The invention includes a polynucleotide sequence encoding a polypeptide sequence having sequence identity to SEQ ID NO: 6569.

The invention further includes a method of screening for an inhibitor of the SARS virus 3CLp protein. In one embodiment, the invention includes a method of screening for an inhibitor of SEQ ID NO: 6569. The invention includes a method of recombinantly expressing the SARS virus 3CLp protein in a host cell. The invention includes a method of recombinantly expressing a polypeptide sequence comprising SEQ ID NO: 6569 or an enzymatically active fragment thereof or a polypeptide sequence having sequence identity thereto. The invention includes a small molecule which inhibits or reduces the proteolytic activity of the SARS virus 3CLp protein. The invention includes a small molecule which inhibits or reduced the proteolytic activity of the polypeptide comprising SEQ ID NO: 6569.

Catalytic residues of the SARS virus 3CLp are identified in FIG. 15 and 16. Specifically, a catalytic histidine and a catalytic cysteine are identified. Such catalytic sites are targets for small molecules which could inhibit or reduce the protease activity of 3CLp. Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6569, wherein said fragment comprises at least one catalytic site. Preferably, the catalytic site is selected from the group consisting of the indicated catalytic histidine and the catalytic cysteine in FIG. 15 and 16. The invention includes a polynucleotide encoding a polypeptide, wherein said polypeptide comprises a fragment of SEQ ID NO: 6569, wherein said fragment comprises at least one catalytic site. Preferably, the catalytic site is selected from the group consisting of the indicated catalytic histidine and the catalytic cysteine.

The invention further includes a method of screening a compound library to identify a small molecule which inhbits a catalytic site of a SARS virus 3CLp. Preferably, the 3CLp comprises SEQ ID NO: 6569, or a fragment thereof, or a sequence having sequence identity thereto. The catalytic site is preferably selected from the group consisting of the indicated catalytic histidine and the catalytic cysteine in FIG. 15 and 16.

The invention includes a small molecule which inhibits the catalytic site of a SARS virus 3CLp. Preferably, the 3CLp comprises SEQ ID NO: 6569, or a fragment thereof, or a sequence having sequence identity thereto. The catalytic site is preferably selected from the group consisting of the indicated catalytic histidine and the catalytic cysteine in FIG. 15 and 16.

Residues of the substrate site of the SARS virus 3CLp are identified in FIG. 15 and 16. Specifically, a substrate site is indicated at a phenylalanine, a tyrosine and a histidine. Such substrate sites are targets for small molecules which could inhibit or reduce the protease activity of 3CLp. Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6569, wherein said fragment comprises at least one substrate site. Preferably, the substrate site is selected from the group consisting of the indicated substrate phenylalanine, tyrosine and histidine in FIG. 15 and 16. The invention includes a polynucleotide encoding a polypeptide, wherein said polypeptide comprises a fragment of SEQ ID NO: 6569, wherein said fragment comprises at least one substrate site. Preferably, the substrate site is selected from the group consisting of the indicated substrate phenylalanine, tyrosine and histidine in FIG. 15 and 16.

The invention further includes a method of screening a compound library to identify a small molecule which blocks a substrate site of a SARS virus 3CLp. Preferably, the 3CLp comprises SEQ ID NO: 6569, or a fragment thereof, or a sequence having sequence identity thereto. The substrate site is preferably selected from the group consisting of the indicated substrate phenylalanine, tyrosine and histidine in FIG. 15 and 16.

The invention includes a small molecule which inhibits the substrate site of a SARS virus 3CLp. Preferably, the 3CLp comprises SEQ ID NO: 6569, or a fragment thereof, or a sequence having sequence identity thereto. The substrate site is preferably selected from the group consisting of the indicated substrate phenylalanine, tyrosine and histidine in FIG. 15 and 16.

The invention further includes a diagnostic kit comprising a polynucleotide encoding a SARS virus 3CLp or a fragment thereof. Preferably, the SARS virus 3CLp comprising SEQ ID NO: 6569 or a fragment thereof or a polypeptide sequence having sequence identity thereto. Preferably, the fragment comprising one or more sites selected from the group consisting of a catalytic site and a substrate site. Preferably, the catalytic site is selected from the group consisting of one or more of the sites identified in FIG. 15 and 16. Preferably, the substrate site is selected from the group consisting of one or more of the sites identified in FIG. 15 and 16.

The invention further comprises a diagnostic kit comprising an antibody specific to a SARS virus 3CLp or a fragment thereof. Preferably, the antibody is specific to the polypeptide comprising SEQ ID NO: 6569 or a fragment thereof or a polypeptide sequence having sequence identity thereto. Preferably, the antibody is specific to one or more sites of a SARS virus 3CLp selected from the group consisting of a catalytic site and a substrate site. Preferably, the catalytic site is selected from the group consisting of one or more of the sites identified in FIG. 15 and 16. Preferably, the substrate site is selected from the group consisting of one or more of the sites identified in FIG. 15 and 16.

The invention includes a polypeptide comprising an amino acid sequence from the sequence shown in FIG. 25. The two amino acid sequences within FIG. 25, separated by a *, are SEQ ID NOS: 7188 & 7189. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to the FIG. 25 translation. The invention includes a fragment of a polypeptide comprising the FIG. 25 sequence. The invention includes a diagnostic kit comprising a polypeptide comprising the FIG. 25 translation, or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding the FIG. 25 translation, or a fragment thereof. The invention includes an immunogenic composition comprising a polypeptide comprising the FIG. 25 translation, or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising the FIG. 25 sequence, or a fragment thereof. The FIG. 25 sequence demonstrates functional homology with ORF1b of coronaviruses.

SEQ ID NO: 7188 is an open reading frame within FIG. 25. The invention includes a polypeptide comprising SEQ ID NO: 7188. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 7188. The invention includes a fragment of a polypeptide comprising SEQ ID NO: 7188. The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 7188, or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding SEQ ID NO: 7188, or a fragment thereof. The invention includes an immunogenic composition comprising a polypeptide comprising SEQ ID NO: 7188, or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 7188, or a fragment thereof.

SEQ ID NO: 7190 is an open reading frame within SEQ ID NO: 7188. The invention includes a polypeptide comprising SEQ ID NO: 7190, a fragment thereof or a polypeptide having sequence identity thereto. The invention further includes a polynucleotide encoding SEQ ID NO: 7190, a fragment thereof or a polypeptide sequence having sequence identity thereto. An example of a polynucleotide encoding SEQ ID NO: 7190 is given as SEQ ID NO: 7191.

SEQ ID NO: 7188 also contains an open reading frame comprising SEQ ID NO: 6042. The invention includes a polypeptide comprising SEQ ID NO: 6042. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 6042. The invention includes a fragment of a polypeptide comprising SEQ ID NO: 6042. The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 6042, or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding SEQ ID NO: 6042, or a fragment thereof. The invention includes an immunogenic composition comprising a polypeptide comprising SEQ ID NO: 6042, or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 6042, or a fragment thereof. SEQ ID NO: 6042 demonstrates functional homology to a coronavirus spike protein.

Predicted transmembrane regions of SEQ ID NO: 6042 are identified below.

Predicted Transmembrane Helices of SEQ ID NO: 6042

The sequence positions in brackets denominate the core region.

Only scores above 500 are considered significant. from to score center Inside to outside helices: 18 found 1 (1) 16 (16) 959 9 233 (237) 257 (252) 905 244 345 (347) 364 (361) 490 354 345 (354) 369 (369) 420 362 497 (497) 513 (513) 239 506 573 (573) 588 (588) 811 580 645 (648) 666 (663) 302 656 690 (696) 714 (711) 428 704 857 (860) 882 (874) 1508 867 1031 (1031) 1046 (1046) 446 1039 1199 (1203) 1219 (1217) 2667 1210 Outside to inside helices: 13 found 1 (1) 17 (17) 684 10 222 (222) 240 (237) 238 229 244 (247) 264 (264) 613 254 349 (355) 369 (369) 314 362 496 (496) 511 (511) 488 503 573 (573) 591 (591) 712 581 650 (652) 666 (666) 474 659 674 (679) 702 (696) 190 686 691 (696) 713 (711) 210 704 866 (868) 886 (886) 1172 876 1198 (1201) 1215 (1215) 3221 1208

SEQ ID NO: 6042, the spike protein, is a surface exposed polypeptide. Recombinant expression of a protein can be hindered by hydrophobic transmembrane regions. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042 wherein one or more of the hydrophobic regions identified above is removed. The invention further includes a polynucleotide encoding such a polypeptide. The invention includes recombinantly expressing the protein in a host cell. Primers for amplifying the gene for spike protein and fragments thereof, such as fragments encoding the soluble ectodomain, include SEQ ID NOS: 9753-9763 (Xiao et al. (2003) Biochem Biophys Res Comm 312:1159-1164).

Further characterization of SEQ ID NO: 6042 is set forth below. PSORT --- Prediction of Protein Localization Sites version 6.4(WWW) SEQ ID NO: 6042 - 1255 Residues Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 2     Position of the most N-terminal TMS: 496 at i = 2 MTOP: membrane topology (Hartmann et al.)     I(middle): 503 Charge diffirence(C − N): 1.0 McG: Examining signal sequence (McGeoch)     Length of UR:  13     Peak Value of UR:  3.28     Net Charge of CR: 0     Discriminant Score:   8.66 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): 5.94     Possible cleavage site: 13 >>> Seems to have a cleavable N-term signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 14 ALOM new cnt: 1 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)     count: 1  value: −12.26  threshold: −2.0     INTEGRAL Likelihood = −12.26  Transmembrane 1202-1218 (1194-1228)     PERIPHERAL Likelihood =  0.16     modified ALOM score:  2.55 >>> Seems to be a Type Ia membrane protein     The cytoplasmic tail is from 1219 to 1255 (37 Residues) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway (14) or uncleavable? Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 14     Uncleavable? Ipos set to: 24 Discrimination of mitochondrial target seq.:     positive (2.18) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway *** Reasoning Step: 2 KDEL   Count: 0 Checking apolar signal for intramitochondrial sorting   (Gavel position 24)  from: 1  to: 10  Score: 8.0 SKL motif (signal for peroxisomal protein):     pos: 964(1255), count: 1 SRL     SKL score (peroxisome): 0.1 Amino Acid Composition Tendency for Peroxisome: 1.37     AAC not from the N-term., score modified Peroxisomal proteins? Status: notclr     AAC score (peroxisome): 0.079 Amino Acid Composition tendency for lysosomal proteins     score: 0.39 Status: notclr GY motif in the tail of typeIa? (lysosomal) Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal  Status: negative (0.00) Type Ia is favored for plasma memb. proteins Checking the NPXY motif.. Checking the YXRF motif.. Checking N-myristoylation.. ----- Final Results ----- plasma membrane --- Certainty = 0.460(Affirmative) <succ> microbody (peroxisome) --- Certainty = 0.171(Affirmative) <succ> endoplasmic reticulum (membrane) --- Certainty = 0.100(Affirmative) <succ> endoplasmic reticulum (lumen) --- Certainty = 0.100(Affirmative) <succ>

SEQ ID NO: 6042 appears to have a N-terminus signaling region, followed by a surface exposed region, followed by a transmembrane region followed by a C-terminus cytoplasmic domain region. Accordingly, the invention includes an immunogenic, surface exposed fragment of SEQ ID NO: 6042. Preferably, said fragment comprises an amino acid sequence which does not include the last 50 amino acids of the C-terminus of SEQ ID NO: 6042. Preferably, said fragment comprises an amino acid sequence which does not include the last 70 amino acids of the C-terminus of SEQ ID NO: 6042. Preferably, said fragment does not include a transdomain region of SEQ ID NO: 6042. Preferably, said fragment does not include a C-terminus cytoplasmic domain of SEQ ID NO: 6042. Preferably, said fragment does not include a N-terminus signal sequence. Preferably, said fragment does not include amino acids 1-10 of the N-terminus of SEQ ID NO: 6042. Preferably, said fragment does not include amino acids 1-14 of the N-terminus of SEQ ID NO: 6042. Two oligopeptide fragments of SEQ ID NO: 6042 that are able to elicit anti-spike antibodies are SEQ ID NOS: 7398 & 7399, as described (with additional C-terminus cysteines) by Xiao et al. (2003) Biochem Biophys Res Comm 312:1159-1164. C-terminal truncations of spike protein, with removal of part of the cytoplasmic region, or removal up to and including the transmembrane region, are described by Yang et al. (2004) Nature 428:561-564.

A variant of SEQ ID NO: 6042 that is included within the invention is SEQ ID NO: 9962. Compared to SEQ ID NO: 6042, this sequence has Ser at residue 581 instead of Ala, and has Phe at residue 1152 instead of Leu.

The spike protein of coronaviruses may be cleaved into two separate chains into S1 and S2. The chains may remain associated together to form a dimer or a trimer. Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 6042 wherein said polypeptide has been cleaved into S1 and S2 domains. The invention further includes a polypeptide comprising SEQ ID NO: 6042 wherein amino acids 1-10, preferably amino acids 1-14 of the N-terminus are removed and further wherein SEQ ID NO: 6042 is cleaved into S1 and S2 domains. Preferably the polypeptide is in the form of a trimer.

The spike protein appears to form an alpha-helical structure in the transmembrane region of the protein, preferably in the S2 domain. This alpha-helical structure is thought to associate with at least two additional spike proteins to form a trimer. Helical or coiled regions of the spike protein are identified below. Predicted coiled-coils of SEQ ID NO: 6042 (spike protein) are at amino acids 900-1005 and 1151-1185 (see FIG. 12).

Accordingly, the invention comprises a polypeptide sequence comprising a fragment of SEQ ID NO: 6042 wherein said fragment includes a coiled region of SEQ ID NO: 6042. Said fragment preferably includes the amino acid sequences selected from the group consisting of amino acid positions 900 to 1005 and amino acid positions 1151 to 1185 of SEQ ID NO: 6042. The invention comprises a polypeptide sequence comprising a fragment of SEQ ID NO: 6042, wherein said fragment does not include a coiled region of SEQ ID NO: 6042. Said fragment preferably includes the amino acid sequences selected from the group consisting of amino acid positions 900 to 1005 and amino acid positions 1151 and 1185 of SEQ ID NO: 6042.

The spike protein is believed to play an integral role in fusion and infection of Coronaviruses with mammalian host cells. Analysis of coronavirus spike proteins as well as similar surface proteins in other viruses has identified at least two structural motifs, typically located within the S2 domain, associated with this fusion event: heptad repeats (HR) and membrane fusion peptides.

At least two 4,3 hydrophobic heptad repeat (HR) domains are typically found in the ectodomain of the S2 domain of Coronaviruses. One heptad repeat region (HR1) is typically located adjacent to a fusion peptide while a second heptad region (HR2) is typically located near the C-terminus of the S2 domain, close to the transmembrane anchor. Heptad repeats are characteristic of coiled-coil structures and the heptad repeats found in viral surface proteins (such as coronavirus spike protein) are thought to form bundled helix structures which are involved in viral entry. See Bosch et al., J. Virology (2003) 77:8801-8811 (FIG. 1B of this reference illustrates an alignment of the HR1 and HR2 regions of five coronaviruses along with SARS, annotated “HCov-SARS”).

Heptad repeats generally contain a repeating structure of seven amino acids, designated a-b-c-d-e-f-g, where hydrophobic sidechains of residues a and d typically form an apolar stripe, and electrostatic interactions are found in residues e and g. Position a is most frequently Leu, Ile or Ala and position d is usually Leu or Ala. Residues e and g are often Glu or Gln, with Arg and Lys also prominent at position g. Charged residues are common to positions b, c and f as these residues may be in contact with solvent. Exceptions to these general parameters are known. For instance Pro residues are sometimes found within the heptad.

The HR1 and HR2 sequences of an MHV strain have been postulated to assemble into a thermostable, oligomeric, alphahelical rold-like complex, with the HR1 and HR2 helices oriented in an antiparallel manner. Id. In this same study, HR2 was asserted to be a strong inhitibor of both virus entry into the cell and cell-cell fusion.

HR1 and HR2 sequences have been identified in the SARS virus genome. The SARS virus HR1 region comprises approximately amino acids 879 to 1005 of SEQ ID NO: 6042 or fragments thereof capable of forming at least one alpha-helical turn. Preferably, said fragments comprise at least 7 (e.g., at least 14, 21, 28, 35, 42, 49 or 56) amino acid residues. SEQ ID NO: 7192, includes amino acids 879 to 1005 of SEQ ID NO: 6042.

A preferred fragment of HR1 comprises amino acid residues 879 to 980 of SEQ ID NO: 6042. This preferred fragment is SEQ ID NO: 7193.

Another preferred fragment of HR1 comprises amino acid residues 901 to 1005 of SEQ ID NO: 6042. This preferred fragment is SEQ ID NO: 7194.

The SARS virus HR2 region comprises approximately amino acids 1144 to 1201 of SEQ ID NO: 6042, or fragments thereof capable of forming at least one alpha-helical turn. Preferably, said fragments comprise at least 7 (e.g., at least 14, 21, 28, 35, 42, 49 or 56) amino acid residues. SEQ ID NO: 7195 includes amino acids 1144 to 1201. A preferred fragment of HR2 comprises amino acids 1144 to 1195 of SEQ ID NO: 6042. This preferred fragment is SEQ ID NO: 7196.

Membrane Fusion peptides sequences within the spike protein are also believed to participate in fusion (and infection) of the virus with a host cell. Fusion peptides generally comprise about 16 to 26 amino acid residues which are conserved within viral families. These Membrane Fusion peptides are relatively hydrophobic and generally show an asymmetric distribution of hydrophobitiy when modeled into an alpha helix. They are also generally rich in alanine and glycine.

At least three hydrophobic Membrane Fusion peptide regions have been identified within coronaviruses (PEP1, PEP2, and PEP3). See, Luo et al., “Roles in Cell-Cell Fusion of Two Conserved Hydrophobic Regions in the Murine Coronavirus Spike Protein”, Virology (1998) 244:483-494. FIG. 1 of this paper shows an alignment of Membrane Fusion peptide sequences of Mouse Hepatitis Viris, Bovine Corona Virus, Feline Infectious Peritonitis Virus, Transmissible Gastroenteritis Virus and Infectious Bronchitis Virus. See also, Bosch et al., “The Coronavirus Spike Protein is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex” Journal of Virology (2003) 77(16):8801-8811.

PEP1 (SEQ ID NO: 7197), PEP2 (SEQ ID NO: 7198) and PEP3 (SEQ ID NO: 7199) sequences within the SARS spike protein have been identified.

The coronavirus spike proteins (and other similar surface viral proteins) are thought to undergo a conformational change upon receptor binding to the target cell membrane. One or more of the hydrophobic Membrane Fusion peptides are thought to become exposed and inserted into the target membrane as a result of this conformational change. The free energy released upon subsequent refolding of the spike protein to its most stable conformation is believed to play a role in the merger of the viral and cellular membranes.

One or more SARS HR sequences, preferably HR2, or a fragment thereof may be used to inhibit viral entry and membrane fusion with a target mammalian host cell. The invention provides a method of inhibiting viral infection comprising administering a composition comprising one or more SARS HR polypeptides or a fragment thereof. Preferably, the composition comprises a SARS HR2 sequence.

In another embodiment, the invention includes a composition comprising a SARS HR1 sequence, or a fragment thereof and a SARS HR2 sequence, or a fragment thereof. The HR1 and HR2 sequences may optionally be associated together in an oligomer. The composition may comprise the intermediate domain sequence between the HR1 and HR2 domains. The use of such an intermediate sequence may facilitate oligomerization or other structural interaction between the HR regions.

HR sequences for use in the invention may be produced recombinantly by methods known in the art. The SARS HR sequences may be modified to facilitate bacterial expression. In particular, the HR sequences may be modified to facilitate transport of the recombinant protein to the surface of the bacterial host cell. For example, leader sequences to a bacterial membrane protein may be added to the N terminus of the recombinant HR sequences. HR sequences for use in the invention may alternatively be produced by chemical synthesis by methods known in the art (see below).

As discussed in more detail later in the specification, Applicants have identified structural similarities between the SARS spike protein and the surface protein of Neisseria meningitidis, NadA (and other similar bacterial adhesion proteins). Another means of facilitating bacterial expression of HR sequences includes the addition of the stalk and/or anchor sequences of a NadA-like protein to the C-terminus of the recombinant HR sequences. Recombinant sequences containing the bacterial anchor sequence may preferably be prepared in outer membrane vesicles (the preparation of which is discussed in more detail later in the application). Recombinant sequences missing the bacterial anchor sequences may be secreted and isolated from the supernatant.

The invention includes a polypeptide sequence comprising a first sequence and a second sequence, wherein said first sequence comprises a leader sequence for a bacterial membrane protein and wherein said second sequence comprises a HR sequence of a coronavirus. Preferably, said first sequence comprises the leader sequence for a bacterial adhesin protein. More preferably, said bacterial adhesion protein is NadA. Preferably said second sequence comprises HR1, HR2 or both. In one embodiment, the second sequence comprises HR1, HR2 and the intermediate domain sequence present in the naturally occrding spike protein. For example, the second sequence may comprise a fragment of a coronavirus spike protein comprising the amino acids starting with the N-terminus of the HR1 region and ending with the C-terminus of the HR2 region.

The invention further includes a polypeptide sequence comprising a first, second, third and fourth sequence, wherein the first sequence comprises a leader sequence for a bacterial membrane protein; wherein said second sequence comprises a HR sequence of a coronavirus; wherein said third sequence comprises a stalk domain of a bacterial adhesion protein; and wherein said fourth sequence comprises an anchor domain of a bacterial adhesion protein. In one embodiment, the first sequence comprising the leader peptide sequence is removed. In another embodiment, the third sequence comprising the stalk domain is removed. In another embodiment, the fourth sequence comprising the anchor domain is removed.

The polypeptide sequences of the above described constructs may be linked together by means known in the art, including, for example, via glycine linkers.

Examples of constructs which may be used in such bacterial expression systems are shown in FIG. 50. Polypeptide sequences of each of the constructs illustrated in FIG. 50 are given as SEQ ID NOS: 7200 to 7206.

7200 Leader NadA (1-29)—HR1 (879-980)—6× gly—HR2 (1144-1195)—stalk+anchor NadA (88-405)

7201 Leader NadA (1-29)—HR1 (879-980)—6× gly—HR2 (1144-1196)—stalk NadA (88-351)

7202 Leader NadA (1-29)—HR1—HR2 (879-1196)—stalk+anchor NadA (88-405)

7203 Leader NadA (1-29)—HR1—HR2 (879-1196)—stalk NadA (88-351)

7204 HR1—HR2 (879-1196)—stalk NadA (88-351)-6× his

7205 Leader NadA (1-29)—HR1—HR2 (879-1196)-anchor NadA (351-405)

7206 Leader NadA (1-29)—HR1—HR2 (879-1196)

Administration of one of more of these Membrane Fusion sequences may also interfere with the ability of a coronavirus to fuse to a host cell membrane. Accordingly, the invention includes an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7197, SEQ ID NO: 7198 and SEQ ID NO: 7199. The invention further includes an isolated polypeptide comprising an amino acid sequence having sequence homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 7197, SEQ ID NO: 7198 and SEQ ID NO: 7199.

Two or more of these SARS Membrane Fusion peptides can be combined together. The invention includes a composition comprising two SARS Membrane Fusion peptides wherein said peptides are selected from at least two of the amino acids selected from the group consisting of SEQ ID NO: 7197, SEQ ID NO: 7198 and SEQ ID NO: 7199, or a sequence having sequence identity thereto.

Two or more of the SARS Membrane Fusion peptides may be linked together. Accordingly, the invention includes a polypeptide comprising a first amino acid sequence and a second amino acid sequence, wherein said first and second amino acid sequences are selected from the group consisting of SEQ ID NO: 7197, SEQ ID NO: 7198 and SEQ ID NO: 7199, or a sequence having sequence identity thereto. Preferably, said first amino acid sequence and said second amino acid sequence are different SARS Membrane Fusion peptides, i.e., they are not the same.

The invention also includes a method of treating or preventing SARS virus infection comprising administering one or more of the SARS Membrane Fusion peptide compositions described above.

As discussed above, the spike protein is capable of forming trimers. The invention further includes a polypeptide comprising SEQ ID NO: 6042 in trimeric form. The invention includes a composition comprising at least polypeptides wherein each polypeptide comprises at least the alpha-helical coiled region of a SARS virus spike protein. Preferably, the spike protein comprises SEQ ID NO: 6042 or a fragment thereof.

The invention further includes a composition comprising a SARS virus spike protein or a fragment thereof wherein said protein is associated with a transmembrane and wherein said fragment comprises the alpha-helical region of the SARS virus spike protein. Preferably, the composition comprises at least three SARS virus spike proteins or a fragment thereof, wherein the fragment comprises the alpha-helical region of the SARS virus spike protein.

The invention further includes an antibody which specifically binds to a trimeric form of SARS virus spike proteins. Preferably, the spike protein comprises SEQ ID NO: 6042 or a fragment thereof. The invention includes an antibody which specifically binds to a trimeric form of SARS virus spike proteins wherein said proteins are associated with a transmembrane.

The invention further includes an antibody which specifically binds to a monomeric form of SARS virus spike protein or a fragment thereof. Preferably, the antibody specifically binds to a monomeric form of SEQ ID NO: 6042 or a fragment thereof.

The invention further includes a small molecule which interferes with or disrupts the coiling of a SARS viral spike protein trimer.

The invention further includes an attenuated SARS virus for use as a vaccine wherein said attenuated virus contains a polynucleotide insertion, deletion or substitution which does not disrupt the trimeric conformation of the SARS virus spike protein. The invention further includes an attenuated SARS virus for use as a vaccine wherein said attenuated virus contains a polynucleotide insertion, deletion or substitution which does not disrupt the alpha-helical formation of the SARS virus spike protein.

The spike protein may be recombinantly produced. In one embodiment, the spike protein is expressed in virus like particles so that the protein is attached to a cell membrane. Such attachment may facilitate presentation of immunogenic epitopes of the spike protein. Preferably, the alpha-helical portion of the spike protein is associated with the cell membrane. Preferably, the spike proteins form a trimer within the transmembrane region of attachment.

Predicted N-glycosylation sites of SEQ ID NO: 6042 are identified below: Jury NGlyc Position Potential agreement result 29 NYTQ SEQ ID NO: 7207 0.7751 (9/9) +++ 65 NVTG SEQ ID NO: 7208 0.8090 (9/9) +++ 109 NKSQ SEQ ID NO: 7209 0.6081 (7/9) + 119 NSTN SEQ ID NO: 7210 0.7039 (9/9) ++ 158 NCTF SEQ ID NO: 7211 0.5808 (7/9) + 227 NITN SEQ ID NO: 7212 0.7518 (9/9) +++ 269 NGTI SEQ ID NO: 7213 0.6910 (9/9) ++ 318 NITN SEQ ID NO: 7214 0.6414 (9/9) ++ 330 NATK SEQ ID NO: 7215 0.6063 (8/9) + 357 NSTF SEQ ID NO: 7216 0.5746 (8/9) + 589 NASS SEQ ID NO: 7217 0.5778 (6/9) + 602 NCTD SEQ ID NO: 7218 0.6882 (9/9) ++ 699 NFSI SEQ ID NO: 7219 0.5357 (7/9) + 783 NFSQ SEQ ID NO: 7220 0.6348 (9/9) ++ 1080 NGTS SEQ ID NO: 7221 0.5806 (7/9) + 1116 NNTV SEQ ID NO: 7222 0.5106 (5/9) + 1176 NESL SEQ ID NO: 7223 0.6796 (9/9) ++

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6042 wherein said fragment comprises one or more of the glycosylation sites identified above (SEQ ID NOS: 7207-7223). The invention further includes a polynucleotide encoding one or more of the fragments identified above. This glycosylation site can be covalently attached to a saccharide. Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6042 wherein said fragment comprises one or more of the glycosylation sites identified above and wherein said polypeptide is glycosylated at one or more of the sites identified above.

Predicted O-glycosylation sites are identified below: Residue No. Potential Threshold Assignment Thr 698 0.8922 0.7696 T Thr 706 0.9598 0.7870 T Thr 922 0.9141 0.7338 T Ser 36 0.8906 0.7264 S Ser 703 0.8412 0.7676 S

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6042 wherein said fragment comprises one or more of the O-glycosylation sites identified above. The invention further includes a polynucleotide encoding one or more of the fragments identified above. The invention further includes a polypeptide comprising a fragment of SEQ ID NO: 6042 wherein said fragment comprises one or more of the O-glycosylation sites identified above and further wherein the polypeptide is covalently bonded to a saccharide at one or more of the included glycosylation sites.

The invention further includes a polypeptide comprising a fragment of SEQ ID NO: 6042 wherein said fragment comprises one or more of the N-glycosylation sites identified above and further wherein said fragment comprises one or more of the O-glycosylation sites identified above.

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6042 wherein said fragment does not include one or more of the glycosylation sites identified above. The invention also includes a polynucleotide encoding such a polypeptide.

Predicted phosphorylation sites of SEQ ID NO: 6042 are Ser-346, Tyr-195, and Tyr-723. Accordingly, the invention comprises a polypeptide comprising a fragment of SEQ ID NO: 6042 wherein said fragment comprises at least ten amino acid residues and wherein said fragment comprises one or more of the amino acids selected from the group consisting of Ser-346, Tyr-195, and Tyr-723. In one embodiment, one or more of the amino acids selected from the group consisting of Ser-346, Tyr-195, and Tyr-723 are phosphorylated.

Expression and functional characterization of the spike glycoprotein has been described by Xiao et al. (2003) Biochem Biophys Res Comm 312:1159-1164.

T-epitopes for SEQ ID NO: 6042 are identified in Table 16. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified as SEQ ID NOS: 8041-8280; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 8041-8280, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6040. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 6040. The invention includes a fragment of a polypeptide comprising SEQ ID NO: 6040. The invention includes a polynucleotide encoding SEQ ID NO: 6040 or a fragment thereof. The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 6040 or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding SEQ ID NO: 6040 or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 6040 or a fragment thereof.

SEQ ID NO: 6040 demonstrates functional homology with a membrane protein of coronaviruses. Predicted transmembrane helices of SEQ ID NO: 6040 are identified below:

Predicted Transmembrane Helices

The sequence positions in brackets denominate the core region.

Only scores above 500 are considered significant. from to score center Inside to outside helices: 3 found 27 (30) 48 (45) 1138 38 137 (139) 153 (153) 486 146 Outside to inside helices: 3 found 28 (31) 45 (45) 819 38 71 (73) 90 (90) 210 81 136 (142) 156 (156) 272 149

The amino acid region with the highest predicted transmembrane helical region is from amino acid position 27 to 48 of SEQ ID NO: 6040. Such transmembrane regions are often difficult to express recombinantly. Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6040 wherein said fragment does not include the amino acid sequence between positions 27 to 48. The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6040 wherein said fragment does not include the amino acid sequence between positions 28 to 45. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

SEQ ID NO: 6040 is predicted to be a hypothetical protein of the SARS virus. A prediction of the protein localization of SEQ ID NO: 6040 is set forth below. SEQ ID NO: 6040 is predicted to be located in one of the following locations: mitochondrial matrix space, microbody (peroxisome), nucleus, and mitochondrial inner membrane. SEQ ID NO: 6040 is predicted to be associated with an organelle inside an infected cell.

Accordingly, SEQ ID NO: 6040 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprises SEQ ID NO: 6040 or a fragment thereof. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6040 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6040 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6040 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6040 from associating with an organelle inside of an infected cell. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6040. PSORT --- Prediction of Protein Localization Sites                   version 6.4(WWW) SEQ ID NO: 6040     163 Residues Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 0 McG: Examining signal sequence (McGeoch)     Length of UR:  9     Peak Value of UR:  1.75     Net Charge of CR: 1     Discriminant Score:   −2.56 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): 1.94     Possible cleavage site: 53 >>> Seems to have no N-terminal signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 1 ALOM new cnt: 0 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)     count: 0  value: 1.32  threshold: −2.0     PERIPHERAL  Likelihood = 1.32     modified ALOM score:  −1.16 Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 156     HRSVTI Discrimination of mitochondrial target seq.:     notclr (0.88) Rule: mitochondrial protein Rule: mitochondrial protein Rule: mitochondrial protein Rule: mitochondrial protein *** Reasoning Step: 2 KDEL   Count: 0 Checking apolar signal for intramitochondrial sorting   (Gavel position 156)  from: 27  to: 44  Score: 5.0 Mitochondrial matrix? Score: 0.36 SKL motif (signal for peroxisomal protein):     pos: 99(163), count: 1 SKL     SKL score (peroxisome): 0.3 Amino Acid Composition Tendency for Peroxisome: −4.28 Peroxisomal proteins? Status: notclr Amino Acid Composition tendency for lysosomal proteins     score: 0.02 Status: notclr Modified score for lysosome: 0.152 Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting     Found: pos: 132 (5) KRKR Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) nuc modified. Score: 0.60 Nuclear Signal Status: notclr (0.30) Checking CaaX motif.. Checking N-myristoylation.. Checking CaaX motif.. ----- Final Results ----- mitochondrial matrix space --- Certainty = 0.480(Affirmative) <succ> microbody (peroxisome) --- Certainty = 0.300(Affirmative) <succ> nucleus --- Certainty = 0.300(Affirmative) <succ> mitochondrial inner membrane --- Certainty = 0.188(Affirmative) <succ>

Predicted N-glycosylation sites of SEQ ID NO: 6040 are identified below. Jury NGlyc Position Potential agreement result 2 NKTG (SEQ ID NO: 7255) 0.7804 (9/9) +++ 106 NLTL (SEQ ID NO: 7256) 0.6123 (7/9) +

Accordingly, the invention comprises a fragment of SEQ ID NO: 6040 wherein said fragment is at least ten amino acids and wherein said fragment comprises one or more of the asparagines from the amino acid positions of SEQ ID NO: 6040 selected from the group consisting of 2 and 106. The invention includes a fragment of SEQ ID NO: 6040 wherein said fragment comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 7255 and SEQ ID NO: 7256. Preferably, the fragment comprises the amino acid sequence NKTG (SEQ ID NO: 7255).

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6040 wherein said fragment does not include one or more of the glycosylation sites identified above. The invention also includes a polynucleotide encoding such a polypeptide.

T-epitopes for SEQ ID NO: 6040 are identified in Table 14. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 7640-7800; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 7640-7800, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus.

The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6041. SEQ ID NO: 6041 demonstrates functional homology with a portion of an ORF 1ab polyprotein. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 6041. The invention includes a fragment of a polypeptide comprising SEQ ID NO: 6041. The invention includes a polynucleotide sequence encoding an amino acid sequence having sequence identity to SEQ ID NO: 6041. The invention includes a polynucleotide encoding a fragment of a polypeptide comprising SEQ ID NO: 6041.

The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 6041 or a fragment therof. The invention includes a diagnostic kit comprising a polynucleotide encoding SEQ ID NO: 6041 or a fragment thereof. The invention includes an immunogenic composition comprising a polypeptide comprising SEQ ID NO: 6041 or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 6041 or a fragment thereof.

The polyproteins of coronaviruses are associated with enzymatic activity. Accordingly, SEQ ID NO: 6041 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprising SEQ ID NO: 6041 or a fragment thereof. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6041 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6041 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6041 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6041 from performing enzymative activity. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6041.

Predicted transmembrane or hydrophobic regions of SEQ ID NO: 6041 are identified below. Although the polyprotein of coronaviruses is proteolytically cleaved into numerous smaller proteins, hydrophobic domains in the polyprotein are known to mediate the membrane association of the replication complex and to be able to dramatically alter the architecture of host cell membranes. Accordingly, the hydrophobic domains of the polyprotein are targets for genetic mutation to develop attenuated SARS virus vaccines. The hydrophobic domains are also targets for small molecule inhibitors of the SARS virus. The hydrophobic domains may also be used to generate antibodies specific to those regions to treat or prevent SARS virus infection.

Possible Transmembrane Helices of SEQ ID NO: 6041

The sequence positions in brackets denominate the core region.

Only scores above 500 are considered significant. from to score center Inside to outside helices: 18 found 234 (234) 254 (250) 1046 241 256 (256) 272 (270) 252 263 319 (319) 334 (334) 227 327 503 (505) 522 (519) 405 512 613 (615) 633 (629) 619 622 677 (679) 703 (696) 467 689 849 (851) 869 (865) 229 858 1080 (1080) 1097 (1094) 306 1087 1147 (1149) 1163 (1163) 354 1156 1557 (1557) 1581 (1577) 817 1567 1954 (1954) 1971 (1971) 832 1964 2369 (2372) 2395 (2387) 300 2379 2513 (2513) 2532 (2529) 690 2522 Outside to inside helices: 14 found 239 (239) 254 (254) 924 247 239 (248) 272 (263) 468 256 311 (314) 334 (328) 267 321 499 (503) 522 (519) 485 512 617 (617) 634 (631) 425 624 849 (853) 872 (872) 572 864 1147 (1147) 1162 (1162) 765 1155 1564 (1564) 1581 (1579) 883 1572 1951 (1951) 1968 (1966) 657 1958 2513 (2522) 2539 (2537) 711 2529

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6041, wherein said fragment comprises an amino acid sequence including one or more of the hydrophobic transmembrane sequences identified above. The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6041 wherein said fragment comprises one or more of the following polypeptide sequences of SEQ ID NO: 6041: 234-254, 613-633, 1557-1581, 1954-1971, 2513-2532, 239-254, 1564-1581, 1951-1968, 2513-2539. Preferably, the fragment comprises one or more of the following polypeptide sequences of SEQ ID NO: 6041: 234-254 and 239-254. The invention also includes polynucleotides encoding each of the polypeptide fragments identified above.

The invention includes an attenuated SARS virus wherein said attenuated SARS virus contains an addition, deletion or substitution in the polynucleotides encoding for one of the hydrophobic domains identified above. The invention also includes a method for creating an attenuated SARS virus comprising mutating a SARS virus by adding, deleting or substituting the viral genome of the SARS virus to alter the coding of one or more of the hydrophobic domains of SEQ ID NO: 6041 identified above.

The invention includes an antibody which specifically identifies one or more of the hydrophobic regions of SEQ ID NO: 6041 identified above. The invention includes a small molecule which binds to, interferes with the hydrophobicity of or otherwise disrupts one or more of the hydrophobic regions of SEQ ID NO: 6041 identified above.

Predicted N-glycosylation sites of SEQ ID NO: 6041 are identified below: Jury NGlyc Position Potential agreement result 571 NLSH (SEQ ID NO: 7257) 0.6598 (8/9) + 835 NTSR (SEQ ID NO: 7258) 0.5762 (7/9) + 958 NVTD (SEQ ID NO: 7259) 0.7494 (9/9) ++ 1113 NISD (SEQ ID NO: 7260) 0.7259 (8/9) + 1205 NSTL (SEQ ID NO: 7261) 0.6296 (9/9) ++ 1460 NVTG (SEQ ID NO: 7262) 0.6844 (9/9) ++ 1685 NHSV (SEQ ID NO: 7263) 0.5181 (5/9) + 2029 NKTT (SEQ ID NO: 7264) 0.5423 (5/9) +

Accordingly, the invention comprises a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6041, wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention comprises a polypeptide comprising a fragment of SEQ ID NO: 6041 wherein said fragment comprises one or more of sequences SEQ ID NOS: 7257-7264. Preferably, the fragment comprises one or more of the sequences SEQ ID NOS: 7257, 7259, 7260, 7261 and 7262. The invention further comprises a polynucleotide encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6041 wherein said fragment does not include one or more of the glycosylation sites identified above. The invention also includes a polynucleotide encoding such a polypeptide.

T-epitopes for SEQ ID NO: 6041 are identified in Table 15. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 7801-8040; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 7801-8040, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus.

The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide sequence SEQ ID NO: 6043 or a fragment thereof. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 6043. The invention includes a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6043 or a fragment thereof.

Predicted transmembrane regions of SEQ ID NO: 6043 are set forth below. from to score center Inside to outside helices: 4 found 41 (41) 56 (56) 1789 49 76 (79) 99 (99) 2142 89 105 (105) 125 (125) 1250 115 Outside to inside helices: 3 found 41 (41) 59 (56) 2053 49 76 (82) 98 (96) 1580 89 103 (105) 125 (123) 1257 115

The amino acid region with the highest predicted transmembrane helical region is from amino acid position 76 to 99 of SEQ ID NO: 6043. Such transmembrane regions are often difficult to express recombinantly. Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6043 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include the amino acids between positions 27 to 48. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

SEQ ID NO: 6043 is predicted to be a hypothetical protein of the SARS virus. A prediction of the protein localization of SEQ ID NO: 6043 is set forth below. SEQ ID NO: 6043 is predicted to be located in one of the following locations: mitochondrial inner membrane, plasma membrane, Golgi body, and mitochondrial intermembrane space. SEQ ID NO: 6043 may be associated with an organelle inside an infected cell.

Accordingly, SEQ ID NO: 6043 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprises SEQ ID NO: 6043 or a fragment thereof. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6043 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6043 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6043 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6043 from associating with an organelle inside of an infected cell. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6043. PSORT --- Prediction of Protein Localization Sites for SEQ ID NO: 6043                     version 6.4(WWW) Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 3     Position of the most N-terminal TMS: 40 at i = 2 MTOP: membrane topology (Hartmann et al.)     I(middle): 47  Charge diffirence(C − N): 3.5 McG: Examining signal sequence (McGeoch)     Length of UR:  12     Peak Value of UR:  1.41     Net Charge of CR: 0     Discriminant Score:   −4.67 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): 3.44     Possible cleavage site: 15 >>> Seems to have no N-terminal signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 1 ALOM new cnt: 2 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)     count: 2  value: −6.90  threshold: −2.0     INTEGRAL Likelihood = −6.90  Transmembrane  83-99 (78-101)     INTEGRAL Likelihood = −5.04  Transmembrane  40-56 (37-60)     PERIPHERAL Likelihood = −0.32     modified ALOM score:  1.48 >>> Likely a Type IIIb membrane protein (Nexo Ccyt) Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 128     MRCWLC Discrimination of mitochondrial target seq.:     notclr (0.76) Rule: mitochondrial protein Rule: mitochondrial protein Rule: mitochondrial protein Rule: mitochondrial protein *** Reasoning Step: 2 Type IIIa or IIIb is favored for ER memb. proteins KDEL   Count: 0 Checking apolar signal for intramitochondrial sorting   (Gavel position 128)  from: 39  to: 56  Score: 11.5 >>> Seems to have an intramitochondrial signal Mitochondrial inner membrane? Score: 0.59 Mitochondrial intermemb.space? Score: 0.22 SKL motif (signal for peroxisomal protein):     pos: 92(274), count: 1 SHL     SKL score (peroxisome):  0.3 Amino Acid Composition Tendency for Peroxisome:  4.78 Peroxisomal proteins? Status: positive Amino Acid Composition tendency for lysosomal proteins     score: 1.16 Status: notclr Type III proteins may be localized at Golgi Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal  Status: negative (0.00) Check the Number of TMSs for typeIII (plasma memb.) Checking N-myristoylation.. ----- Final Results ----- mitochondrial inner membrane --- Certainty = 0.664(Affirmative) <succ> plasma membrane --- Certainty = 0.600(Affirmative) <succ> Golgi body --- Certainty = 0.400(Affirmative) <succ> mitochondrial intermembrane space --- Certainty = 0.362(Affirmative) <succ>

Predicted N— and O-glycosylation sites of SEQ ID NO: 6043 are identified below. Jury NGlyc Position Potential agreement result 227 NATF (SEQ ID NO: 7265) 0.6328 (7/9) + Residue No. Potential Threshold Assignment Thr 28 0.9095 0.6280 T Thr 32 0.8740 0.6595 T Thr 34 0.9058 0.6655 T Thr 170 0.6816 0.6600 T Thr 267 0.9240 0.5779 T Thr 268 0.7313 0.5708 T Thr 269 0.9859 0.5583 T Thr 270 0.8023 0.5492 T Ser 27 0.6930 0.6091 S Ser 252 0.6457 0.5977 S

Accordingly, the invention comprises a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6043, wherein said fragment comprises the N-glycosylation sites or O-glycosylation sites identified above. The invention comprises a polypeptide comprising a fragment of SEQ ID NO: 6043 wherein said fragment comprises one or more of the N-glycosylation sites or O-glycosylation sites identified above. The invention further comprises a polynucleotide encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6043 wherein said fragment does not include one or more of the glycosylation sites identified above. The invention also includes a polynucleotide encoding such a polypeptide.

T-epitopes for SEQ ID NO: 6043 are identified in Table 17. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 8281-8486; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 8281-8486, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6044. The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6044 or a sequence having sequence identity to SEQ ID NO:206. The invention includes a polynucleotide encoding SEQ ID NO: 6044.

SEQ ID NO: 6044 is identified as a hypothetical protein. Predicted hydrophobic or transmembrane regions of SEQ ID NO: 6044 are identified below: from to score center Inside to outside helices: 3 found 1 (1) 17 (15) 891 8 47 (47) 66 (63) 221 56 Outside to inside helices: 4 found 1 (4) 21 (19) 599 11

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6044 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include the amino acids between positions 1 to 19. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

SEQ ID NO: 6044 is predicted to be a hypothetical protein of SARS virus. A prediction of the protein localization of SEQ ID NO: 6044 is set forth below. SEQ ID NO: 6044 is predicted to be located in one of the following locations: nucleus, mitochondrial matrix, lysosome (lumen), and microbody (peroxisome). SEQ ID NO: 6044 may be associated with an organelle inside an infected cell.

Accordingly, SEQ ID NO: 6044 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprises SEQ ID NO: 6044 or a fragment thereof The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6044 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6044 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6044 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6044 from associating with an organelle inside of an infected cell. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6044. PSORT --- Prediction of Protein Localization Sites for SEQ ID NO: 6044                 version 6.4(WWW) 154 Residues Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 0 McG: Examining signal sequence (McGeoch)     Length of UR:  7     Peak Value of UR:  1.06     Net Charge of CR: 1     Discriminant Score:   −7.97 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): −3.28     Possible cleavage site: 34 >>> Seems to have no N-terminal signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 1 ALOM new cnt: 0 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)     count: 0  value:  1.43  threshold: −2.0     PERIPHERAL  Likelihood = 1.43     modified ALOM score:  −1.19 Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 151     FRKKQV Discrimination of mitochondrial target seq.:     notclr (−0.46) *** Reasoning Step: 2 KDEL   Count: 0 Checking apolar signal for intramitochondrial sorting   (Gavel position 151)  from: 46  to: 50  Score: 5.0 Mitochondrial matrix? Score: 0.36 SKL motif (signal for peroxisomal protein):     pos: −1(154), count: 0 Amino Acid Composition Tendency for Peroxisome: 0.61 Peroxisomal proteins? Status: notclr     AAC score (peroxisome): 0.149 Amino Acid Composition tendency for lysosomal proteins     score: 0.81 Status: notclr Modified score for lysosome: 0.231 Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting     Found: pos: 134 (3) KHKK Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus)     Found: pos: 136 (3) KK VSTNLCTHSF RKKQV Final Robbins Score (nucleus): 0.60 Checking the RNA binding motif (nucleus or cytoplasm) nuc modified.  Score: 0.90 Nuclear Signal  Status: positive (0.70) Checking CaaX motif.. Checking N-myristoylation.. Checking CaaX motif.. ----- Final Results ----- nucleus --- Certainty = 0.880(Affirmative) <succ> mitochondrial matrix space --- Certainty = 0.360(Affirmative) <succ> lysosome (lumen) --- Certainty = 0.231(Affirmative) <succ> microbody (peroxisome) --- Certainty = 0.149(Affirmative) <succ>

One predicted O-glycosylation site of SEQ ID NO: 6044 is identified at residue 4: Residue No. Potential Threshold Assignment Thr 4 0.6839 0.6484 T

Accordingly, the invention comprises a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6044, wherein said fragment comprises the O-glycosylation site identified above. The invention further comprises a polynucleotide encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6044 wherein said fragment does not include one or more of the glycosylation sites identified above. The invention also includes a polynucleotide encoding such a polypeptide.

T-epitopes for SEQ ID NO: 6044 are identified in Table 18. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 8487-8665; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 8487-8665, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide sequence comprising SEQ ID NO: 6045. The invention includes a polypeptide sequence comprising an amino acid sequence having sequence identity to SEQ ID NO: 6045. The invention includes a polypeptide sequence comprising a fragment of SEQ ID NO: 6045. The invention includes a polynucleotide sequence encoding any of these polypeptides.

SEQ ID NO: 6045 demonstrates functional homology with the envelope or small membrane protein of coronaviruses. The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 6045 or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide encoding SEQ ID NO: 6045 or a fragment thereof. The invention includes an immunogenic composition comprising SEQ ID NO: 6045 or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 6045 or a fragment thereof.

Predicted transmembrane regions of SEQ ID NO: 6045 are identified below: from to score center Inside to outside helices: 1 found 17 (19) 33 (33) 2881 26 Outside to inside helices: 1 found 17 (17) 34 (34) 2981 27

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6045 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include the amino acids between positions 17 to 34. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides. In one embodiment, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6045 wherein said fragment does not include amino acid residues 1-34 of SEQ ID NO: 6045.

Predicted protein Localization Site of SEQ ID NO: 6045 is below. PSORT --- Prediction of Protein Localization Sites for SEQ ID NO: 6045                         version 6.4(WWW) Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 2     Position of the most N-terminal TMS: 17 at i = 1 MTOP: membrane topology (Hartmann et al.)     I(middle): 24  Charge diffirence(C-N): 2.0 McG: Examining signal sequence (McGeoch)     Length of UR: 29     Peak Value of UR: 3.40     Net Charge of CR: −2     Discriminant Score: 13.07 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): 4.37     Possible cleavage site: 32 ... positive value of mtop ... >>> Seems to have an uncleavable N-term signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 1 ALOM new cnt: 1 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)     count: 1  value: −15.12  threshold: −2.0     INTEGRAL Likelihood = −15.12  Transmembrane  17-33 (8-44)     PERIPHERAL Likelihood =  0.47     modified ALOM score:  3.12 >>> Seems to be a Type Ib (Nexo Ccyt) membrane protein     The cytoplasmic tail is from 34 to 76 (44 Residues) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway (6) or uncleavable? Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 6     Uncleavable? Ipos set to: 16 Discrimination of mitochondrial target seq.:     notclr (0.19) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway *** Reasoning Step: 2 > Relative position of the end of the tail: 44% Memb.protein with uncleavable signl is often at ER KDEL   Count: 0 Checking apolar signal for intramitochondrial sorting   (Gavel position 16)  from: 70  to: 99  Score: 21.5 >>> Seems to have an intramitochondrial signal SKL motif (signal for peroxisomal protein):     pos: −1(76), count: 0 Amino Acid Composition Tendency for Peroxisome: −4.11 Peroxisomal proteins? Status: negative Amino Acid Composition tendency for lysosomal proteins     score: 0.68 Status: notclr Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal  Status: negative (0.00) Check cytoplasmic tail for typeIb (plasma memb.) Checking the NPXY motif.. Checking the YXRF motif.. Checking N-myristoylation.. ----- Final Results ----- plasma membrane --- Certainty = 0.730(Affirmative) <succ> endoplasmic reticulum (membrane) --- Certainty = 0.640(Affirmative) <succ> endoplasmic reticulum (lumen) --- Certainty = 0.100(Affirmative) <succ> outside --- Certainty = 0.100(Affirmative) <succ>

Predicted N-glycosylation sites of SEQ ID NO: 6045 are identified at residues 48 and 66: Jury NGlyc Position Potential agreement result 48 NVSL 0.6514 (9/9) ++ (SEQ ID NO: 7266) 66 NSSE 0.5880 (7/9) + (SEQ ID NO: 7267)

Accordingly, the invention comprises a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6045, wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention comprises a polypeptide comprising a fragment of SEQ ID NO: 6045 wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention further comprises a polynucleotide encoding one or more of the polypeptides identified above.

The invention includes a polypeptide comprising a fragment of SEQ ID NO: 6045 wherein said fragment does not include one or more of the glycosylation sites identified above. The invention also includes a polynucleotide encoding such a polypeptide.

T-epitopes for SEQ ID NO: 6045 are identified in Table 19. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 8666-8820; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 8666-8820, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide sequence comprising SEQ ID NO: 6046. The invention includes polypeptide sequences comprising an amino acid sequence having sequence identity to SEQ ID NO: 6046. The invention includes a polypeptide sequence comprising a fragment of SEQ ID NO: 6046. The invention includes a polynucleotide encoding one of these polypeptides.

SEQ ID NO: 6046 has functional homology with a matrix protein of a coronavirus. The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 6046 or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide encoding SEQ ID NO: 6046 or a fragment thereof. The invention includes an immunogenic composition comprising SEQ ID NO: 6046 or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 6046 or a fragment thereof.

Predicted transmembrane regions of SEQ ID NO: 6046 are identified below. from to score center Inside to outside helices: 3 found 21 (21) 38 (36) 2412 29 51 (53) 69 (69) 2645 60 74 (82) 96 (96) 2464 89 Outside to inside helices: 3 found 18 (21) 38 (38) 2363 28 52 (52) 67 (67) 2363 60 76 (76) 95 (92) 2605 84

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6046 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include the amino acids between positions selected from the group consisting of 18 to 38, 52 to 67 and 76 to 95. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

Predicted protein localization of SEQ ID NO: 6046 is set forth below. PSORT --- Prediction of Protein Localization Sites                     version 6.4(WWW) Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 3     Position of the most N-terminal TMS: 21 at i = 1 MTOP: membrane topology (Hartmann et al.)     I(middle): 28  Charge diffirence(C − N): 6.0 McG: Examining signal sequence (McGeoch)     Length of UR:  1     Peak Value of UR:  3.16     Net Charge of CR: −3     Discriminant Score:   2.21 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): 4.29     Possible cleavage site: 39 ... positive value of mtop ... >>> Seems to have an uncleavable N-term signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 1 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)     count: 3  value:  −7.64  threshold: 0.5     INTEGRAL Likelihood = −7.64  Transmembrane  21-37 (18-39)     INTEGRAL Likelihood = −7.59  Transmembrane  50-66 (43-72)     INTEGRAL Likelihood = −5.04  Transmembrane  79-95 (72-99)     PERIPHERAL Likelihood =   2.38     modified ALOM score:  2.13 >>> Likely a Type IIIb membrane protein (Nexo Ccyt) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway (2) or uncleavable? Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 2     Uncleavable? Ipos set to: 12 Discrimination of mitochondrial target seq.:     negative (−4.16) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway *** Reasoning Step: 2 Type IIIa or IIIb is favored for ER memb. proteins Memb.protein with uncleavable signl is often at ER KDEL   Count: 0 Checking apolar signal for intramitochondrial sorting SKL motif (signal for peroxisomal protein):     pos: −1(221), count: 0 Amino Acid Composition Tendency for Peroxisome:  5.01 Peroxisomal proteins? Status: notclr Amino Acid Composition tendency for lysosomal proteins     score: 2.30  Status: positive Type III proteins may be localized at Golgi Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal  Status: negative (0.00) Check the Number of TMSs for typeIII (plasma memb.) Checking N-myristoylation.. ----- Final Results ----- endoplasmic reticulum (membrane) --- Certainty = 0.685(Affirmative) <succ> plasma membrane --- Certainty = 0.640(Affirmative) <succ> Golgi body --- Certainty = 0.460(Affirmative) <succ> endoplasmic reticulum (lumen) --- Certainty = 0.100(Affirmative) <succ>

One predicted N-glycosylation sites of SEQ ID NO: 6046 is identified at residue 4:

Prediction of N-glycosylation Sites Jury NGlyc Position Potential agreement result 4 NGTI 0.8430 (9/9) +++ (SEQ ID NO: 7268)

Accordingly, the invention comprises a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6046, wherein said fragment comprises the N-glycosylation site identified above. The invention further comprises a polynucleotide encoding one or more of the polypeptides identified above.

The invention further comprises a polypeptide comprising a fragment of amino acid sequence SEQ ID NO: 6046, wherein said fragment does not include the N-glycosylation site identified above. The invention includes a polynucleotide encoding such a fragment.

A variant of SEQ ID NO: 6046 that is included within the invention is SEQ ID NO: 9963. Compared to SEQ ID NO: 6046, this sequence has Val at residue 72 instead of Ala.

T-epitopes for SEQ ID NO: 6046 are identified in Table 20. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 8821-9018; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 8821-9018, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide sequence comprising SEQ ID NO: 6047 or a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted transmembrane regions of SEQ ID NO: 6047 are identified below. from to score center Inside to outside helices: 2 found  7 (10) 29 (27) 729 17 21 (24) 41 (41) 640 34 Outside to inside helices: 2 found 4 (4) 22 (19) 874 12 22 (24) 41 (41) 499 31

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6047 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include the amino acids between positions selected from the group consisting of 4 to 22 and 22 to 41. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

SEQ ID NO: 6047 is predicted to be a hypothetical protein of SARS virus. A prediction of the protein localization of SEQ ID NO: 6047 is set forth below. SEQ ID NO: 6047 is predicted to be located in one of the following locations: plasma membrane, endoplasmic reticulum, Golgi body, and microbody (peroxisome). SEQ ID NO: 6047 may be associated with an organelle inside an infected cell or with viral entry to a host cell.

Accordingly, SEQ ID NO: 6047 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprises SEQ ID NO: 6047 or a fragment thereof. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6047 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6047 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6047 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6047 from associating with an organelle inside of an infected cell or interacting with a host cell membrane. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6047. Predicted protein localization of SEQ ID NO: 6047 is set forth below. PSORT --- Prediction of Protein Localization Sites                     version 6.4(WWW) Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 1     Position of the most N-terminal TMS: 2 at i = 1 MTOP: membrane topology (Hartmann et al.)     I(middle): 9  Charge diffirence(C − N): 0.5 McG: Examining signal sequence (McGeoch)     Length of UR:  6     Peak Value of UR:  3.08     Net Charge of CR: 0     Discriminant Score:   5.12 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): −4.45     Possible cleavage site: 34 >>> Seems to have an uncleavable N-term signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 1 ALOM new cnt: 1 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)     count: 1  value: −2.44  threshold: −2.0     INTEGRAL Likelihood = −2.44  Transmembrane  2-18 (1-20)     PERIPHERAL Likelihood =  1.22     modified ALOM score:  0.59 >>> Seems to be a Type II (Ncyt Cexo) membrane protein     The cytoplasmic tail is from 1 to 1 (1 Residues) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway (5) or uncleavable? Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 5     Uncleavable? Ipos set to: 15 Discrimination of mitochondrial target seq.:     notclr (1.48) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway *** Reasoning Step: 2 Relative position of the cytoplasmic tail: 1%     Larger value (>30%) is favared for ER memb. proteins Memb.protein with uncleavable signl is often at ER KDEL   Count: 0 Checking apolar signal for intramitochondrial sorting   (Gavel position 15)  from: 64  to: 93  Score: 30.0 >>> Seems to have an intramitochondrial signal SKL motif (signal for peroxisomal protein):     pos: −1(63), count: 0 Amino Acid Composition Tendency for Peroxisome: 1.91 Peroxisomal proteins? Status: notclr     AAC score (peroxisome): 0.161 Amino Acid Composition tendency for lysosomal proteins     score: 0.04 Status: notclr Checking the consensus for Golgi Checking the consensus for Golgi Checking the cytoplasmic tail of type II (Golgi) Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal  Status: negative (0.00) Check mitochondrial signal for typeII (plasma memb.) Type II is favored for plasma memb. proteins Checking the NPXY motif.. Checking the YXRF motif.. Checking N-myristoylation.. ----- Final Results ----- plasma membrane --- Certainty = 0.685(Affirmative) <succ> endoplasmic reticulum (membrane) --- Certainty = 0.640(Affirmative) <succ> Golgi body --- Certainty = 0.370(Affirmative) <succ> microbody (peroxisome) --- Certainty = 0.161(Affirmative) <succ>

T-epitopes for SEQ ID NO: 6047 are identified in Table 21. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 9019-9131; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 9019-9131, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6048, a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted transmembrane regions of SEQ ID NO: 6048 are identified below. from to score center Inside to outside helices: 2 found 3 (3) 18 (18) 1857 10 100 (100) 117 (115) 2904 107 Outside to inside helices: 2 found 1 (1) 15 (15) 1299 8 100 (100) 117 (115) 3009 107

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6048 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. Preferably, the fragment does not include the amino acids between positions selected from the group consisting of 1 to 15 and 100 to 117. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

SEQ ID NO: 6048 is predicted to be a hypothetical protein of SARS virus. A prediction of the protein localization of SEQ ID NO: 6048 is set forth below. SEQ ID NO: 6048 is predicted to be located in one of the following locations: plasma membrane, lysosome (membrane), microbody (peroxisome), and endoplasmic reticulum (membrane). SEQ ID NO: 6048 may be associated with an organelle inside an infected cell or may interact with a host cell plasma membrane during viral entry to the host cell.

Accordingly, SEQ ID NO: 6048 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprises SEQ ID NO: 6048 or a fragment thereof. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6048 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6048 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6048 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6048 from associating with an organelle inside of an infected cell or prevents the polypeptide from associating with the cell membrane of a host cell. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6048. Predicted protein localization of SEQ ID NO: 6048 is set forth below. PSORT --- Prediction of Protein Localization Sites                     version 6.4(WWW) Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 2     Position of the most N-terminal TMS: 3 at i = 2 MTOP: membrane topology (Hartmann et al.)     I(middle): 10  Charge diffirence(C − N): −2.5 McG: Examining signal sequence (McGeoch)     Length of UR:  13     Peak Value of UR:  3.38     Net Charge of CR: 1     Discriminant Score:   10.02 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): 2.56     Possible cleavage site: 15 >>> Seems to have a cleavable N-term signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 16 ALOM new cnt: 2 ** thrshld change to −2 Cleavable signal was detected in ALOM?: 1B ALOM: finding transmembrane regions (Klein et al.)     count: 1  value: −14.75  threshold: −2.0     INTEGRAL Likelihood = −14.75  Transmembrane 101-117 (95-120)     PERIPHERAL Likelihood =  6.63     modified ALOM score:  3.05 >>> Seems to be a Type Ia membrane protein     The cytoplasmic tail is from 118 to 122 (5 Residues) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway (15) or uncleavable? Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 15     Uncleavable? Ipos set to: 25 Discrimination of mitochondrial target seq.:     notclr (0.73) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway *** Reasoning Step: 2 KDEL Count: 0 Checking apolar signal for intramitochondrial sorting   (Gavel position 25)  from: 3  to: 12  Score: 8.5 SKL motif (signal for peroxisomal protein):     pos: −1(122), count: 0 Amino Acid Composition Tendency for Peroxisome: 2.46     AAC not from the N-term., score modified Peroxisomal proteins? Status: notclr     AAC score (peroxisome): 0.115 Amino Acid Composition tendency for lysosomal proteins     score: −0.40  Status: negative GY motif in the tail of typeIa? (lysosomal) Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal  Status: negative (0.00) Type Ia is favored for plasma memb. proteins Checking the NPXY motif.. Checking the YXRF motif.. Checking N-myristoylation.. Checking GPI anchor.. >>> Seems to be GPI-anchored (0.85) ----- Final Results ----- plasma membrane --- Certainty = 0.919(Affirmative) <succ> lysosome (membrane) --- Certainty = 0.200(Affirmative) <succ> microbody (peroxisome) --- Certainty = 0.115(Affirmative) <succ> endoplasmic reticulum (membrane) --- Certainty = 0.100(Affirmative) <succ>

T-epitopes for SEQ ID NO: 6048 are identified in Table 22. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 9132-9308; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 9132-9308, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6049, a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted transmembrane or hydrophobic regions of SEQ ID NO: 6049 are identified below. from to score center Inside to outside helices: 1 found 13 (13) 30 (28) 3532 20 Outside to inside helices: 1 found  9 (11) 29 (26) 3395 19

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6049 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

SEQ ID NO: 6049 is predicted to be a hypothetical protein of SARS virus. A prediction of the protein localization of SEQ ID NO: 6049 is set forth below. SEQ ID NO: 6049 is predicted to be located in one of the following locations: outside, microbody (peroxisome), endoplasmic reticulum (membrane) and endoplasmic reticulum (lumen). The highest ranking indicates that SEQ ID NO: 6049 is located on the outside of a cell. Accordingly, SEQ ID NO: 6049 may be a surface exposed protein.

Accordingly, SEQ ID NO: 6049 may be used in an immunogenic composition to raise an immune response against the SARS virus. It also may be used to generate antibodies specific to the SARS virus. Such antibodies may be used in a method of treatment or prevention of a SARS virus infection. Such antibodies may further be used in a diagnostic test to identify the presence or absence of SARS virus in a biological sample.

The invention includes a polypeptide comprises SEQ ID NO: 6049 or a fragment thereof. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6049 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6049 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6049 in a host cell. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6049. Predicted protein localization of SEQ ID NO: 6049 is set forth below. PSORT --- Prediction of Protein Localization Sites                     version 6.4(WWW) Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)     count: 1     Position of the most N-terminal TMS: 11 at i = 1 MTOP: membrane topology (Hartmann et al.)     I(middle): 18 Charge diffirence(C − N): −2.0 McG: Examining signal sequence (McGeoch)     Length of UR:  24     Peak Value of UR:  3.69     Net Charge of CR: −2     Discriminant Score:   13.56 GvH: Examining signal sequence (von Heijne)     Signal Score (−3.5): 0.52     Possible cleavage site: 25 >>> Seems to have a cleavable N-term signal seq. Amino Acid Composition of Predicted Mature Form:    calculated from 26 ALOM new cnt: 1 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 1B ALOM: finding transmembrane regions (Klein et al.)     count: 0  value: 14.80  threshold: −2.0     PERIPHERAL  Likelihood = 14.80     modified ALOM score: −3.86 Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway (2) or uncleavable? Gavel: Examining the boundary of mitochondrial targeting seq.      motif at: 2     Uncleavable? Ipos set to: 12 Discrimination of mitochondrial target seq.:     notclr (1.42) Rule: vesicular pathway Rule: vesicular pathway Rule: vesicular pathway *** Reasoning Step: 2 KDEL   Count: 0 Number of Potential N-glycosylation Sites: 0 Out: score 0.800 Checking apolar signal for intramitochondrial sorting   (Gavel position 12)  from: 44  to: 73  Score: 30.0 >>> Seems to have an intramitochondrial signal SKL motif (signal for peroxisomal protein):     pos: −1(44), count: 0 Amino Acid Composition Tendency for Peroxisome: 9.47     AAC not from the N-term., score modified Peroxisomal proteins? Status: notclr     AAC score (peroxisome): 0.320 Amino Acid Composition tendency for lysosomal proteins     score: −6.47  Status: negative Number of NX(S/T) motif: 0 Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal  Status: negative (0.00) Checking CaaX motif.. Checking N-myristoylation.. Checking CaaX motif.. ----- Final Results ----- outside --- Certainty = 0.820(Affirmative) <succ> microbody (peroxisome) --- Certainty = 0.320(Affirmative) <succ> endoplasmic reticulum (membrane) --- Certainty = 0.100(Affirmative) <succ> endoplasmic reticulum (lumen) --- Certainty = 0.100(Affirmative) <succ>

T-epitopes for SEQ ID NO: 6049 are identified in Table 23. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 9309-9437; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 9309-9437, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide comprising SEQ ID NO: 6050 or a fragment thereof or an amino acid sequence having sequence identity thereto. Predicted transmembrane or hydrophobic regions are identified below. from to score center Inside to outside helices: 1 found 13 (15) 32 (30) 558 23 Outside to inside helices: 1 found 16 (16) 30 (30) 364 23

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6050 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

SEQ ID NO: 6050 is predicted to be a hypothetical protein of SARS virus. A prediction of the protein localization of SEQ ID NO: 6050 is set forth below. SEQ ID NO: 6050 is predicted to be located in one of the following locations: lysosome (lumen), mitochondrial matrix space, mitochondrial inner membrane, and mitochondrial intermembrane space. SEQ ID NO: 6050 may be associated with an organelle inside an infected cell during the viral replication cycle.

Accordingly, SEQ ID NO: 6050 is a target for screening of chemical inhibitors to the SARS virus. The invention includes a polypeptide comprises SEQ ID NO: 6050 or a fragment thereof. The invention includes a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6050 or a fragment thereof. The invention includes a method of screening SEQ ID NO: 6050 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6050 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6050 from associating with an organelle inside of an infected cell or prevents the polypeptide from associating with the cell membrane of a host cell. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6050. Predicted protein localization of SEQ ID NO: 6050 is set forth below. PSORT --- Prediction of Protein Localization Sites version 6.4(WWW) MYSEQ    84 Residues Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)  count: 0 McG: Examining signal sequence (McGeoch)  Length of UR:  3  Peak Value of UR:  1.46  Net Charge of CR: 2  Discriminant Score:   −5.73 GvH: Examining signal sequence (von Heijne)  Signal Score (−3.5): −0.12  Possible cleavage site: 29 >>> Seems to have no N-terminal signal seq. Amino Acid Composition of Predicted Mature Form:  calculated from 1 ALOM new cnt: 0 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)  count: 0 value: 8.43 threshold: −2.0  PERIPHERAL Likelihood = 8.43  modified ALOM score: −2.59 Gavel: Examining the boundary of mitochondrial targeting seq.   motif at: 61  ARCWYL Discrimination of mitochondrial target seq.:  positive (1.66) Rule: mitochondrial protein Rule: mitochondrial protein Rule: mitochondrial protein Rule: mitochondrial protein *** Reasoning Step: 2 KDEL Count: 0 Checking apolar signal for intramitochondrial sorting  (Gavel position 61) from: 52 to: 58 Score: 6.0 Mitochondrial matrix? Score: 0.38 SKL motif (signal for peroxisomal protein):  pos: −1(84), count: 0 Amino Acid Composition Tendency for Peroxisome: 1.47 Peroxisomal proteins? Status: notclr  AAC score (peroxisome): 0.263 Amino Acid Composition tendency for lysosomal proteins  score: 2.86 Status: positive Modified score for lysosome: 0.850 Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus) Checking the RNA binding motif (nucleus or cytoplasm) Nuclear Signal Status: negative (0.00) Checking CaaX motif.. Checking N-myristoylation.. Checking CaaX motif.. ----- Final Results ----- lysosome (lumen) --- Certainty = 0.850(Affirmative) < succ> mitochondrial matrix space --- Certainty = 0.544(Affirmative) < succ> mitochondrial inner membrane --- Certainty = 0.266(Affirmative) < succ> mitochondrial intermembrane space --- Certainty = 0.266(Affirmative) < succ>

One predicted N-glycosylation sites of SEQ ID NO: 6050 is identified at residue 43: Jury NGlyc Position Potential agreement result 43 NVTI 0.6713 (9/9) ++ (SEQ ID NO: 7269)

Accordingly, the invention comprises a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6050 wherein said fragment comprises the N-glycosylation site identified above. The invention further comprises a polynucleotide encoding one or more of the polypeptides identified above.

The invention further comprises a polypeptide comprising a fragment of amino acid sequence SEQ ID NO: 6050 wherein said fragment does not include the N-glycosylation site identified above. The invention includes a polynucleotide encoding such a fragment.

T-epitopes for SEQ ID NO: 6050 are identified in Table 24. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 9438-9538; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 9438-9538, or a polynucleotide encoding such a polypeptide.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a polypeptide sequence comprising SEQ ID NO: 6051 or a fragment thereof or an amino acid sequence having sequence identity thereto. The invention includes a polypeptide sequence comprising SEQ ID NO: 6052 or a fragment thereof or an amino acid sequence having sequence identity thereto.

SEQ ID NO: 6051 and SEQ ID NO: 6052 demonstrate functional homology with a nucleocapsid protein of a coronavirus. The invention includes a diagnostic kit comprising a polypeptide comprising SEQ ID NO: 6051, SEQ ID NO: 6052 or a fragment thereof. The invention includes a diagnostic kit comprising a polynucleotide encoding SEQ ID NO: 6051, SEQ ID NO: 6052 or a fragment thereof. The invention includes an immunogenic composition comprising SEQ ID NO: 6051, SEQ ID NO: 6052 or a fragment thereof. The invention includes an antibody which recognizes a polypeptide comprising SEQ ID NO: 6051, SEQ ID NO: 6052 or a fragment thereof.

SEQ ID NO: 6051 is predicted to be phosphorylated at Ser-79; Thr-92; Ser-106; Thr-116; Thr-142; Ser-184; Ser-188; Ser-202; Ser-236; Thr-248; Ser-251; Ser-256; Thr-377. Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6051 wherein said fragment includes one or more of the amino acid residues of SEQ ID NO: 6051 selected from the group consisting of Ser-79; Thr-92; Ser-106; Thr-116; Thr-142; Ser-184; Ser-188; Ser-202; Ser-236; Thr-248; Ser-251; Ser-256; Thr-377. The invention further includes a polypeptide comprising a fragment of SEQ ID NO: 6051 wherein said fragment does not include one or more of the amino acid residues of SEQ ID NO: 6051 selected from the group consisting of Ser-79; Thr-92; Ser-106; Thr-116; Thr-142; Ser-184; Ser-188; Ser-202; Ser-236; Thr-248; Ser-251; Ser-256; Thr-377. Two further useful fragments of the N protein (e.g. for immunoassay) are SEQ ID NOS: 9783 & 9784, which are lysine-rich and can be used to distinguish the SARS virus from other coronaviruses.

Predicted transmembrane regions of SEQ ID NO: 6051 are identified below. from to score center Inside to outside helices: 1 found 304 (304) 323 (319) 495 312 Outside to inside helices: 2 found 304 (304) 319 (319) 597 312

Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 6051 wherein said fragment does not include one or more of the hydrophobic amino acid sequences identified above. The invention also includes a polynucleotide sequence encoding any of the above-identified polypeptides.

Predicted protein localization of SEQ ID NO: 6051 is set forth below. SEQ ID NO: 6051 is predicted to be localized near the nucleus, lysosome (lumen), mitochondrial matrix space, and microbody (peroxisome). The highest ranking is for localization near the nucleus. Coronavirus nucleocapsid proteins are known to bind to viral RNA. Coronavirus nucleocapsid proteins are also thought to be important for cell mediated immunity. Accordingly, the invention includes a polynucleotide comprising SEQ ID NO: 6051. The invention further includes a viral vector or particle suitable for in vivo delivery of the polynucleotide sequence comprising a SARS virus nucleocapsid polynucleotide sequence or a fragment thereof. In one embodiment, the polynucleotide comprises SEQ ID NO: 6051 or a fragment thereof. The invention further includes a method for eliciting a cell mediated immune response comprising delivering a polynucleotide encoding a SARS virus nucleocapsid protein or a fragment thereof to a mammal. In one embodiment, the polynucleotide comprising SEQ ID NO: 6051 or a fragment thereof.

The invention further includes a method of screening SEQ ID NO: 6051 for an inhibitor. The invention includes the recombinant expression of SEQ ID NO: 6051 in a host cell. The invention includes a small molecule which prevents the polypeptide of SEQ ID NO: 6051 from binding to SARS virus RNA during viral replication. The invention includes a fusion protein wherein said fusion protein comprises SEQ ID NO: 6051. Predicted protein localization of SEQ ID NO: 6051 is set forth below. PSORT --- Prediction of Protein Localization Sites version 6.4(WWW) Species classification: 4 *** Reasoning Step: 1 Preliminary Calculation of ALOM (threshold: 0.5)  count: 0 McG: Examining signal sequence (McGeoch)  Length of UR: 3  Peak Value of UR: 0.19  Net Charge of CR: 0  Discriminant Score:  −15.98 GvH: Examining signal sequence (von Heijne)  Signal Score (−3.5): −6.36  Possible cleavage site: 58 >>> Seems to have no N-terminal signal seq. Amino Acid Composition of Predicted Mature Form:  calculated from 1 ALOM new cnt: 0 ** thrshld changed to −2 Cleavable signal was detected in ALOM?: 0B ALOM: finding transmembrane regions (Klein et al.)  count: 0 value: 5.04 threshold: −2.0  PERIPHERAL Likelihood = 5.04  modified ALOM score: −1.91 Gavel: Examining the boundary of mitochondrial targeting seq.   motif at: 17  PRITFG Discrimination of mitochondrial target seq.:  negative (−3.97) *** Reasoning Step: 2 KDEL Count: 0 Checking apolar signal for intramitochondrial sorting Mitochondrial matrix? Score: 0.10 SKL motif (signal for peroxisomal protein):  pos: −1(399), count: 0 Amino Acid Composition Tendency for Peroxisome: 0.04 Peroxisomal proteins? Status: notclr  AAC score (peroxisome): 0.072 Amino Acid Composition tendency for lysosomal proteins  score: 0.96 Status: notclr Modified score for lysosome: 0.246 Checking the amount of Basic Residues (nucleus) Checking the 4 residue pattern for Nuclear Targeting  Found: pos: 256 (4) KKPR  Found: pos: 372 (5) KKKK Checking the 7 residue pattern for Nuclear Targeting Checking the Robbins & Dingwall consensus (nucleus)  Found: pos: 372 (3) KK KKTDEAQPLP QRQKK Found: pos: 373 (3) KK KTDEAQPLPQ RQKKQ Final Robbins Score (nucleus): 0.80 Checking the RNA binding motif (nucleus or cytoplasm) nuc modified. Score: 0.90 Nuclear Signal Status: positive (0.90) Checking CaaX motif.. Checking N-myristoylation.. Checking CaaX motif.. ----- Final Results ----- nucleus --- Certainty = 0.980(Affirmative) < succ> lysosome (lumen) --- Certainty = 0.246(Affirmative) < succ> mitochondrial matrix space --- Certainty = 0.100(Affirmative) < succ> microbody (peroxisome) --- Certainty = 0.072(Affirmative) < succ>

Predicted N-glycosylation sites of SEQ ID NO: 6051 are identified below. Jury NGlyc Position Potential agreement result  48 NNTA 0.6879 (9/9) ++ (SEQ ID NO: 7270) 270 NVTQ 0.7684 (9/9) +++ (SEQ ID NO: 7271)

Residue No. Potential Threshold Assignment Thr 166 0.8547 0.6439 T Thr 367 0.5575 0.5403 T Thr 394 0.8217 0.5821 T

Accordingly, the invention comprises a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 6051 wherein said fragment comprises one or more of the N-glycosylation sites identified above. The invention further comprises a polynucleotide encoding one or more of the polypeptides identified above.

The invention further comprises a polypeptide comprising a fragment of amino acid sequence SEQ ID NO: 6051 wherein said fragment does not include one or more of the N-glycosylation sites identified above. The invention includes a polynucleotide encoding such a fragment.

T-epitopes for SEQ ID NO: 6052 are identified in Table 25. The invention includes a polypeptide for use as an antigen, wherein the polypeptide comprises: (a) an amino acid sequence selected from the group consisting of the T-epitope sequences identified in SEQ ID NOS: 9539-9752; (b) an amino acid sequence having sequence identity to an amino acid sequence of (a). The invention further comprising a polynucleotide sequence encoding the polypeptides of (a) or (b). The invention further comprising a method of expression or delivery of such polynucleotides through viral vectors and/or viral particles. The invention further comprises a polypeptide comprising two or more of the T-epitope sequences identified in SEQ ID NOS: 9539-9752, or a polynucleotide encoding such a polypeptide.

A variant of SEQ ID NO: 6052 that is included within the invention is SEQ ID NO: 9964. Compared to SEQ ID NO: 6052, this sequence has Ile at residue 54 instead of Thr.

The use as an antigen is preferably a use: (1) as a T-cell antigen; (2) for generating a complex between a class I MHC protein (e.g. a class I HLA) and a fragment of said antigen; (3) as an antigen for raising a cell-mediated immune response; and/or (4) as an antigen for raising a CTL response. The use preferably protects or treats disease and/or infection caused by a SARS virus. The invention provides the use of a polypeptide in the manufacture of a medicament for immunising a mammal (typically a human) against SARS viral infection wherein the polypeptide is as defined above.

The invention provides a method of raising an immune response in a mammal (typically a human), comprising the step of administering to the mammal a polypeptide as defined above, wherein said immune response is a cell-mediated immune response and, preferably, a CTL response. The immune response is preferably protective or therapeutic.

The invention includes a composition comprising a SARS virus nucleocapsid protein or a fragment thereof and further comprising a SARS virus membrane protein or a fragment thereof. The composition may further comprising one or more adjuvants discussed below.

The invention further includes a composition comprising a polypeptide comprising SEQ ID NO: 6051 or a fragment thereof or a sequence having sequence identity thereto and further comprising a polypeptide comprising SEQ ID NO: 6040, or a fragment thereof or a sequence having sequence identity thereto. Such composition may be used, for instance, in a vaccine. Such composition may further comprise one or more adjuvants discussed below.

The invention includes a composition comprising a SARS virus nucleocapsid protein or a fragment thereof and a SARS virus spike protein or a fragment thereof. In one embodiment the nucleocapsid protein comprises a polypeptide sequence comprising SEQ ID NO: 6051 or a fragment thereof or a sequence having sequence identity thereto. In one embodiment, the spike protein comprises a polynucleotide comprising SEQ ID NO: 6042 or a fragment thereof or a sequence having sequence identity thereto. The composition may further comprise one or more of the adjuvants discussed below.

The invention further includes a composition comprising antibodies specific to a SARS virus nucleocapsid protein and comprising antibodies specific to a SARS virus spike protein. In one embodiment the antibody is specific to a nucleocapsid protein comprises a polypeptide sequence comprising SEQ ID NO: 6051 or a fragment thereof or a sequence having sequence identity thereto. In one embodiment, the antibody is is specific to a spike protein comprises a polynucleotide comprising SEQ ID NO: 6042 or a fragment thereof or a sequence having sequence identity thereto.

The invention further includes polynucleotide sequences, and fragments thereof, of a SARS virus which are conserved among coronaviruses, and polypeptides encoded thereby. Such conserved sequences can be identified in the alignments shown in FIG. 7. Such conserved sequences may be used in the vaccines of the invention or in the diagnostic reagents, kits and methods of the invention.

The invention further includes polynucleotide sequences, and fragments thereof, of a SARS virus which are specific to SARS virus and not shared with coronaviruses. Such SARS specific sequences are also identified as SEQ ID NOS: 6040, 6043, 6044, 6047, 6048, 6049 and 6050. Such SARS specific sequences may be used in the vaccines of the invention or in the diagnostic reagents, kits and methods of the invention.

The invention also includes polynucleotide sequences which can be used as probes or primers for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified in SEQ ID NOS: 6076-6265 (Table 5). The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in SEQ ID NOS: 6076-6265.

The invention also includes polynucleotide sequences which can be used as probes or primers for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified in SEQ ID NOS: 6266-6343 (Table 6). The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in SEQ ID NOS: 6266-6343.

The invention also includes polynucleotide sequences which can be used as probes or primers for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified in SEQ ID NOS: 6344-6392 (Table 7). The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in SEQ ID NOS: 6344-6392.

The invention also includes polynucleotide sequences which can be used as probes or primers for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified in SEQ ID NOS: 6393-6559 (Tables 8 & 9). The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in SEQ ID NOS: 6393-6559.

The invention also includes polynucleotide sequences which can be used as probes or primers for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer and probe sequences identified in SEQ ID NOS: 6560-6568. The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in SEQ ID NOS: 6560-6568.

The invention includes a polypeptide sequence comprising any one of even-numbered SEQ ID NOS: 7272-7290, or a fragment thereof, or a sequence having sequence identity thereto. The invention further includes a polynucleotide sequence encoding any one of even-numbered SEQ ID NOS: 7272-7290, or a fragment thereof, or a sequence having sequence identity thereto. Examples of such polynucleotide sequences are odd-numbered SEQ ID NOS: 7273-7291.

The invention includes a polynucleotide sequence comprising an intergenic sequence which is common to each open reading frame of the SARS virus. The SARS virus is thought to use this sequence to signal translation of the open reading frame. The intergenic sequence comprises a 10 mer SEQ ID NO: 7292, or optionally a hexamer SEQ ID NO: 7293. When the virus transcribes its positive (+) RNA strand to (−) RNA strand, the virus replicating structure uses the (−) strand template to transcribe nucleotides at the 5′ end prior to the first intergenic sequence, followed by the intergenic sequence, followed by the selected open reading frame. The virus then creates multiple mRNAs comprising the 5′ end, the intergenic sequence and coding sequence. For more details on Nidovriales replication (including Coronavirus) see e.g., Ziebuhr et al., “Virus-encoded proteinases and proteolytic processing in the Nidovirales”, Journal of General Virology 81:853-879 (2000), incorporated herein by reference in its entirety.

The invention comprising a polynucleotide sequence comprising SEQ ID NO: 7292 or the complement thereof. The invention comprising a polynucleotide sequence comprising SEQ ID NO: 7293 or the complement thereof. The invention further comprises a polynucleotide sequence comprising nucleotides from the 5′ end of the SARS viral genome, or its reverse complement, and farther comprising an intergenic sequence or its reverse complement. The polynucleotide may further comprise one or more of the SARS virus open reading frames. Examples of polynucleotide sequences comprising nucleotides from the 5′ end of the SARS virus genome followed by the intergenic sequence are SEQ ID NOS: 7294-7301.

The invention includes a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301, or a fragment thereof, or a sequence having sequence identity thereto. In one embodiment, the polynucleotide does not consist entirely of a known SARS virus sequence.

The SARS virus intergenic sequence can be used to create a RNAi molecule. Such a SARS virus specific RNAi molecule can be used to treat SARS virus infection. The invention includes a RNAi molecule comprising a double stranded RNA molecule wherein one RNA strand comprises a sequence selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301, or a fragment thereof. Preferably, said RNA strand comprises a sequence selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. Preferably, the other RNA strand comprises the reverse complement of the first strand or a polynucleotide sequence which hybridizes to the first strand.

The invention includes the use of RNAi in a method of treatment for SARS virus infection comprising administering to a mammal an effective amount of the si RNA molecule. Preferably, the RNAi molecule comprises the molecule described above. Further discussion of the RNAi applications of the intergenic sequence is included in section IV of the specification below.

The invention also includes the use of a SARS virus antisense nucleotide sequence, preferably antisense directed to the SARS virus intergenic sequence. Such an antisense sequence may be used in the treatment of a subject infected with the SARS virus. The antisense of the SARS virus intergenic sequence can be designed to bind to the SARS viral polynucleotides to block access of the viral replication machinery to the intergenic sequence. Such an antisense sequence may also be used to identify the presence or absence of a SARS virus in a biological sample. The antisence can itself be labeled or the antisense associated with viral polynucleotides can be detected by means known in the art.

Antisense nucleic acids are designed to specifically bind to RNA, resulting in the formation of RNA-DNA or RNA-RNA hybrids, with an arrest of DNA replication, reverse transcription or messenger RNA translation. Antisense polynucleotides based on a selected sequence can interfere with expression of the corresponding gene. Antisense polynucleotides will bind and/or interfere with the translation of the corresponding mRNA.

The invention also includes the use of the intergenic region with a ribozyme.

Trans-cleaving catalytic RNAs (ribozymes) are RNA molecules possessing endoribonuclease activity. Ribozymes are specifically designed for a particular target, and the target message must contain a specific nucleotide sequence. They are engineered to cleave any RNA species site-specifically in the background of cellular RNA. The cleavage event renders the mRNA unstable and prevents protein expression. Importantly, ribozymes can be used to inhibit expression of a gene of unknown function for the purpose of determining its function in an in vitro or in vivo context, by detecting the phenotypic effect.

One commonly used ribozyme motif is the hammerhead, for which the substrate sequence requirements are minimal. Design of the hammerhead ribozyme is disclosed in Usman et al., Current Opin. Struct. Biol. (1996) 6:527-533. Usman also discusses the therapeutic uses of ribozymes. Ribozymes can also be prepared and used as described in Long et al., FASEB J. (1993) 7:25; Symons, Ann. Rev. Biochem. (1992) 61:641; Perrotta et al., Biochem. (1992) 31:16-17; Ojwang et al., Proc. Natl. Acad. Sci. (USA) (1992) 89:10802-10806; and U.S. Pat. No. 5,254,678. Ribozyme cleavage of HIV-I RNA is described in U.S. Pat. No. 5,144,019; methods of cleaving RNA using ribozymes is described in U.S. Pat. No. 5,116,742; and methods for increasing the specificity of ribozymes are described in U.S. Pat. No. 5,225,337 and Koizumi et al., Nucleic Acid Res. (1989) 17:7059-7071. Preparation and use of ribozyme fragments in a hammerhead structure are also described by Koizumi et al., Nucleic Acids Res. (1989) 17:7059-7071. Preparation and use of ribozyme fragments in a hairpin structure are described by Chowrira & Burke, Nucleic Acids Res. (1992) 20:2835. Ribozymes can also be made by rolling transcription as described in Daubendiek & Kool, Nat. Biotechnol. (1997) 15(3):273-277.

The hybridizing region of the ribozyme may be modified or may be prepared as a branched structure as described in Horn & Urdea, Nucleic Acids Res. (1989) 17:6959-67. The basic structure of the ribozymes may also be chemically altered in ways familiar to those skilled in the art, and chemically synthesized ribozymes can be administered as synthetic oligonucleotide derivatives modified by monomeric units. In a therapeutic context, liposome mediated delivery of ribozymes improves cellular uptake, as described in Birikh et al., Eur. J. Biochem. (1997) 245:1-16.

Therapeutic and functional genomic applications of ribozymes proceed beginning with knowledge of a portion of the coding sequence of the gene to be inhibited. In the present invention, the target sequence preferably comprises the intergeneic sequence of the SARS virus. Preferably, the sequence is selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. A target cleavage site is selected in the target sequence, and a ribozyme is constructed based on the 5′ and 3′ nucleotide sequences that flank the cleavage site. Preferably, the 5′ nucleotide sequence includes the 5′ untranslated region of the SARS virus. The ribozyme may then further be constructed from one or more of the polynucleotide sequences selected from the group consisting of SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301.

Antisense treatment of HIV infection is described in the following references, each of which is incorporated herein by reference in their entirety. (antisense RNA complementary to the mRNA of gag, tat, rev, env) (Sezakiel et al., 1991, J. Virol. 65:468-472; Chatterjee et al., 1992, Science 258:1485-1488; Rhodes et al., 1990, J. Gen. Virol. 71:1965. Rhodes et al., 1991, AIDS 5:145-151; Sezakiel et al., 1992, J. Virol. 66:5576-5581; Joshi et al., 1991, J. Virol. 65:5524-5530).

The invention includes the use of decoy RNA to disrupt the SARS virus replication and life cycle. Methods of making and using such decoy RNA for treatment of a viral infection are known in the art. The invention includes delivery of genes encoding, for example, the SARS virus intergenic sequence, to infected cells. Preferably, the sequence comprises one or more of the sequences selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301. Preferably, the sequence comprises one or more of the sequences selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. Preferably, the sequence comprises SEQ ID NO: 7293.

In the present invention, delivery of intergenic sequence which is not linked to the SARS virus open reading frames disrupts the translation process of the viral RNA and decreases the production of vial proteins. Similar methods of treatment for HIV viral infection have been described. The following references discuss the use of decoy RNA of HIV TAR or RRE for treatment of HIV infection. Each of these references is incorporated herein by reference in their entirety. (Sullenger et al., 1990, Cell 63:601-608; Sullenger et al., 1991, J. Virol. 65:6811-6816; Lisziewicz et al., 1993, New Biol. 3:82-89; Lee et al., 1994, J. Virol. 68:8254-8264), ribozymes (Sarver et al., 1990, Science 247:1222-1225; Wecrasinghe et al., 1991, J. Virol. 65:5531-5534; Dropulic et al., 1992, J. Virol. 66:1432-1441; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA. 89:10802-10806; Yu et al., 1993, Proc. Natl. Acad. Sci. USA. 90:6340-6344; Yu et al., 1995, Proc. Natl. Acad. Sci. USA. 92:699-703; Yamada et al., 1994, Gene Therapy 1:38-45).

The invention includes the use of the SARS virus intergenic sequence in diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. Such diagnostic reagents, kits, and methods are further discussed in Section II of the specification.

The invention includes a pair of primers for amplifying a SARS polynucleotide sequence comprising (i) a first primer comprising a sequence which is substantially identical to a portion of a sequence selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301 and (ii) a second primer comprising a sequence which is substantially complementary to a portion of a sequence selected from the group consisting of the sequence SEQ ID NO: 1 and the sequence SEQ ID NO: 2, such that the primer pair (i) and (ii) defines a template sequence within a sequence from the group consisting of the sequence SEQ ID NO: 1 and the sequence SEQ ID NO: 2. Preferably, the (i) first primer comprises a sequence which is substantially identical to a portion of a sequence selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. Preferably, the (i) first primer comprises a sequence which is substantially identical to a portion of the sequence of SEQ ID NO: 7293. The amplicon defined by said first and second primers is preferably between 50 and 250 nucleotides in length. The primers may optionally be labeled to facilitate their detection. Methods and compositions for use in labeling primers are discussed further in the application in Section III.

The invention further includes a pair of primers for amplifying a SARS polynucleotide sequence comprising (i) a first primer comprising a sequence which is substantially identical to a portion of the complement of a portion of a sequence selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301 and (ii) a second primer comprising a sequence which is substantially complementary to a portion of the complement of a sequence selected from the group consisting of the sequence SEQ ID NO: 1 and the sequence SEQ ID NO: 2, such that the primer pair defines a template sequence within a sequence selected from the group consisting of the sequence SEQ ID NO: 1 and the sequence SEQ ID NO: 2. The amplicon defined by said first and second primers is preferably between 50 and 250 nucleotides in length. The primers may optionally be labeled to facilitate their detection. Methods and compositions for use in labeling primers are discussed further in the application in Section III.

The invention includes a kit comprising (i) a first primer comprising a sequence which is substantially identical to a portion of a sequence selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301 and (ii) a second primer comprising a sequence which is substantially complementary to a portion of a sequence selected from the group consisting of the sequence SEQ ID NO: 1 and the sequence SEQ ID NO: 2, such that the primer pair (i) and (ii) defines a template sequence within a sequence from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. Preferably, the (i) first primer comprises a sequence which is substantially identical to a portion of a sequence selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. Preferably, the (i) first primer comprises a sequence which is substantially identical to a portion of the sequence of SEQ ID NO: 7293. The primers may optionally be labeled to facilitate their detection. Methods and compositions for use in labeling primers are discussed further in the application in Section III.

Other preferred kits comprise (i) a first primer comprising a sequence which is substantially identical to a portion of the complement of a portion of a sequence selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301 and (ii) a second primer comprising a sequence which is substantially complementary to a portion of the complement of a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, such that the primer pair defines a template sequence within a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

The invention further includes an attenuated SARS virus for use as a vaccine wherein the intergenic region has been mutated to reduce expression of the viral structural or nonstructural proteins. The attenuated SARS virus may comprises one or more additions, deletions or insertion in one or more of the intergenic regions of the viral genome. Preferably, the attenuated SARS virus comprises an addition, deletion or insertion in one or more occurrences of the sequence selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. Preferably, the addition, deletion or insertion occurs in one or more occurrences of SEQ ID NO: 7293.

The invention further comprises a small molecule which inhibits binding or association of the SARS viral replication machinery, such as a ribonucleoprotein, with the intergenic region of the viral genome. Preferably, the small molecule inhibits binding or association of the SARS viral machinery with a sequence selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. Preferably, the small molecule inhbiits binding or association of the SARS viral machinery with SEQ ID NO: 7293. The invention further includes a method of screening for a small molecule for treatment of SARS viral infection comprising using an assay to identify a small molecule which interferes with the association of the SARS viral replication machinery with the intergenic region of the SARS viral genome.

The invention further provides a novel SARS polynucleotide sequence SEQ ID NO: 9968. All six reading frames of this 690 mer sequence are shown in FIG. 113. The constituent amino acid sequences from FIG. 113, having at least 4 amino acids, are listed as SEQ ID NOS: 9969 to 10032.

Accordingly the invention includes a polynucleotide sequence comprising SEQ ID NO: 9968. It also provides polynucleotide sequences having sequence identity to SEQ ID NO: 9968. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 80%, 85%, 88%, 90%, 92%, 95%, 99% or more).

The invention includes an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 9968, including the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 9969 to 10032. Preferably, the amino acid sequence comprises SEQ ID NO: 9997 or comprises SEQ ID NO: 9998.

The invention also provides amino acid sequences having sequence identity to an amino acid sequence encoded by SEQ ID NO: 9968. The invention provides amino acids having sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO^(S): 9969 to 10032. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 80%, 85%, 88%, 90%, 92%, 95%, 99% or more).

A portion of SEQ ID NO: 9968 matches with approximately 98% identity to a previously published SARS polynucleotide sequence, commonly referred to as “BNI-1” (SEQ ID NO: 10033). BNI-1 was sequenced at Bernhard Nocht Institute for Tropical Medicine, National Reference Center for Tropical Infectious Diseases in Hamburg, Germany. The BNI-1 sequence was published on the WHO website on Apr. 4, 2003 at http://www.who.int/csr/sars/primers/en and in Dorsten et al., “Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome”, New England Journal of Medicine, published online at http://www.nejm.org on Apr. 10, 2003. Both references are incorporated herein by reference in their entirety. The six reading frames of this 302 mer sequence are shown in FIG. 114 (see also FIG. 129). The constituent amino acid sequences from FIG. 114, having at least 4 amino acids, are listed as SEQ ID NO_(S): 10034 to 10065. An alignment of SEQ ID NO: 10034 with SEQ ID NO: 9997 is shown in FIG. 130.

The invention provides for polynucleotide sequences comprising fragments of SEQ ID NO: 9968. In one embodiment, the fragment does not consist entirely of SEQ ID NO: 10033 or of a known coronavirus.

The invention provides for amino acid sequences comprising fragments of an amino acid sequence encoded by SEQ ID NO: 9968. In one embodiment, the fragment does not consist entirely of an amino acid sequence encoded by SEQ ID NO: 10033 or a known coronavirus.

The invention provides for amino acids comprising fragments of an amino acid sequence selected from the group consisting of SEQ ID NO^(S): 9969 to 10032. In one embodiment, the fragment does not consist entirely of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 10033 or a known coronavirus.

Approximately 100 nucleotides at the 5′ end of SEQ ID NO: 9968 do not match any portion of the BNI-1 polynucleotide sequence (SEQ ID NO: 10033). This unmatched portion is set forth as SEQ ID NO: 10066. The invention thus further provides a polynucleotide comprising the sequence comprising SEQ ID NO: 10066, polynucleotide sequences having sequence identity to SEQ ID NO: 10066, or polynucleotide sequences comprising fragments of SEQ ID NO: 10066.

The invention further comprises an amino acid sequence encoded by SEQ ID NO: 10066, an amino acid sequence having sequence identity to an amino acid sequence encoded by SEQ ID NO: 10066, or an amino acid sequence comprising fragments of an amino acid sequence encoded by SEQ ID NO: 10066. Preferably, the amino acid sequence comprises SEQ ID NO: 10067.

SEQ ID NO: 9997/9998 demonstrates homology with the a region of pol 1ab of several coronaviruses. FIG. 115 shows an alignment of SEQ ID NO^(S): 9997/9998 to amino acid sequences for pol 1ab of bovine coronavirus (SEQ ID NO: 10068), avian infectious bronchitis virus (SEQ ID NO: 10069) and murine hepatitis virus (SEQ ID NO: 10070). A consensus amino acid sequence of SEQ ID NO^(S): 9997/9998, SEQ ID NO: 10068, SEQ ID NO: 10069, and SEQ ID NO: 10070 is shown in the bottom row of the alignment in FIG. 115 (e.g. SEQ ID NO: 10071).

As shown in FIG. 113, the polynucleotide sequence encoding SEQ ID NO: 9997 has a stop codon after codon 205, between SEQ ID NO^(S): 9997 and 9998. Optionally, the stop codon can be removed and the amino acid sequence continued (SEQ ID NO: 10072). Accordingly, the invention provides for an amino acid sequence comprising SEQ ID NO: 9997 and/or SEQ ID NO: 9998, or SEQ ID NO: 10072, and further comprising an amino acid sequence encoding for the C-terminus of a coronavirus pol 1ab gene or a fragment thereof.

As shown in FIG. 115, SEQ ID NO^(S): 10068, 10069, 10070 and 10071 contain amino acids prior to the N-terminus of SEQ ID NO: 9997. The invention also provides for an amino acid sequence comprising SEQ ID NO: 9997 and further comprising an amino acid sequence encoding for the N-terminus of a coronavirus pol1ab protein or a fragment thereof.

The pol1ab sequences on FIG. 115 contain a coding region indicated on the schematic of FIG. 117 by a “*”. In FIG. 115, the beginning of this genomic region is designated by the arrow crossing in front of amino acid 6080 of the consensus sequence SEQ ID NO: 10071. The end of this genomic region is designated by the arrow crossing in front of amino acid 6604 of the consensus sequence. The invention provides for an amino acid sequence comprising SEQ ID NO: 9997 and/or SEQ ID NO: 9998, or SEQ ID NO: 10072, and further comprising a first amino sequence prior to the N-terminus of said SEQ ID NO: 9997 and/or SEQ ID NO: 9998, or SEQ ID NO: 10072, wherein said first amino acid sequence has homology to an N-terminus sequence of a known coronavirus pol 1ab “*” protein or a fragment thereof.

The invention further provides for an amino acid sequence comprising SEQ ID NO: 9997 and SEQ ID NO: 9998, wherein the stop codon after SEQ ID NO: 9971 is removed (i.e. SEQ ID NO: 10072), and further comprising a second amino acid sequence following the C terminus of SEQ ID NO: 9998, wherein said second amino acid sequence is homologous with a C terminus of a known coronavirus pol 1ab “*” protein or a fragment thereof.

Examples of such proteins are shown aligned in FIG. 118, and are SEQ ID NO^(S): 10073 to 10077. SEQ ID NO: 10073 comprises SEQ ID NO: 9997 and further comprises amino acids prior to the N-terminus and subsequent to the C-terminus from the pol 1ab “*” protein of avian infectious bronchitis virus. SEQ ID NO: 10074 comprises SEQ ID NO: 9997 and further comprises amino acids prior to the N-terminus and subsequent to the C-terminus from the pol1ab “*” protein of bovine coronavirus. SEQ ID NO: 10075 comprises SEQ ID NO: 9997 and further comprises amino acids prior to the N-terminus and subsequent to the C-terminus from the pol 1ab “*” protein of murine hepatitis virus. SEQ ID NO: 10076 comprises SEQ ID NO: 9997 and further comprises amino acids prior to the N-terminus and subsequent to the C-terminus from the consensus of the pol1ab “*” protein of avian infectious bronchitis virus, bovine coronavirus, and murine hepatitis virus (FIG. 115). SEQ ID NO: 10077 comprises the consensus sequence of SEQ ID NOS: 10073 to 10076.

The invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO^(S): 10073, 10074, 10075, 10076 and 10077. The invention further includes an amino acid sequence comprising fragments of an amino acid sequence selected from the group consisting of SEQ ID NO^(S): 10073, 10074, 10075, 10076 and 10077. The invention further comprises an amino acid sequence with sequence identity to a sequence selected from the group consisting of SEQ ID NO^(S): 10073, 10074, 10075, 10076 and 10077.

The invention comprises polynucleotides encoding for the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 10073, 10074, 10075, 10076 and 10077. The invention comprises polynucleotides having sequence identity to polynucleotides encoding for the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 10073, 10074, 10075, 10076 and 10077. The invention comprises fragments of polynucleotides encoding SEQ ID NO^(S): 10073, 10074, 10075, 10076 and 10077.

As shown in FIG. 113, SEQ ID NO: 9968 includes a sequence that encodes SEQ ID NO: 10020 followed by a stop codon, giving a C-terminus threonine (Thr) residue. The corresponding sequence from an amino acid sequence encoded by BNI-1 is SEQ ID NO: 10078, which continues past the C-terminus of SEQ ID NO: 10020. Accordingly, the invention includes a protein comprising amino acid sequence SEQ ID NO: 10020 or an amino acid sequence having sequence identity to SEQ ID NO: 10020 or an amino acid sequence comprising a fragment of SEQ ID NO: 10020, wherein the C-terminus residue of said protein is a threonine. Preferably, the C-terminus of said protein is -ST. Still more preferably, the C-terminus of said protein is -EST. The invention also includes a protein comprising amino acid sequence SEQ ID NO: 10078 or an amino acid sequence having sequence identity to SEQ ID NO: 10078 or an amino acid sequence comprising a fragment of SEQ ID NO: 10078, wherein the C-terminus residue of said protein is Thr. Preferably, the C-terminus of said protein is -ST. Still more preferably, the C-terminus of said protein is -EST.

SEQ ID NO: 9968 also encodes a 54 mer amino acid sequence SEQ ID NO: 10015. The polynucleotide encoding SEQ ID NO: 10015 encodes two stop codons at its C-terminus (FIG. 113). The corresponding region from the BNI-1 sequence does not contain this 54 mer. Accordingly, the invention includes a protein comprising amino acid sequence SEQ ID NO: 10015, or an amino acid sequence having sequence identity to SEQ ID NO: 10015 or an amino acid sequence comprising a fragment of SEQ ID NO: 10015. The invention further includes a polypeptide comprising SEQ ID NO: 10015 and further comprising a first amino acid sequence prior to the N-terminus of SEQ ID NO: 10015.

SEQ ID NO: 9968 encodes the amino acid sequence SEQ ID NO: 9969. The polynucleotide sequence contains a stop codon at the C-terminus of SEQ ID NO: 9969. Accordingly, the invention includes a protein comprising amino acid sequence SEQ ID NO: 9969, or an amino acid sequence having sequence identity to SEQ ID NO: 9969. The invention further includes a polypeptide comprising SEQ ID NO: 9969 and further comprising a first amino acid sequence prior to the N-terminus of SEQ ID NO: 9969. The invention further includes a polypeptide comprising the sequence SEQ ID NO: 10079.

SEQ ID NO: 9968 encodes amino acid sequence QRT (FIG. 113), followed by a stop codon. Accordingly, the invention includes a protein comprising amino acid sequence QRT. The invention further includes a polypeptide comprising amino acid sequence QRT and further comprising a first amino acid sequence prior to the N-terminus of the sequence QRT.

SEQ ID NO: 9968 encodes amino acid sequence SEQ ID NO: 10022, followed by a stop codon at its C-terminus. Accordingly, the invention includes a protein comprising amino acid sequence SEQ ID NO: 10022, or an amino acid sequence having sequence identity to SEQ ID NO: 10022. The invention further includes a polypeptide comprising SEQ ID NO: 10022 and further comprising a first amino acid sequence prior to the N-terminus of SEQ ID NO: 10022.

SEQ ID NO: 9968 encodes amino acid sequence SEQ ID NO: 10027. Within the SEQ ID NO: 10027 coding sequence there are at least three start codons, identified with underlining in FIG. 119. The open reading frame indicated by the first start codon is SEQ ID NO: 10081. The open reading frame indicated by the second start codon is SEQ ID NO: 10082. The open reading frame indicated by the third start codon is SEQ ID NO: 10083.

The invention provides a novel SARS polynucleotide sequence SEQ ID NO: 10084. All six reading frames of this 1463 mer sequence are shown in FIG. 120 (see also FIG. 122). The constituent amino acid sequences from FIG. 120, having at least 4 amino acids, are listed as SEQ ID NOS: 10085 to 10209 (see FIGS. 120A to 120F).

The invention includes a polynucleotide sequence comprising SEQ ID NO: 10084. The invention also provides polynucleotide sequences having sequence identity to SEQ ID NO: 10084. The invention also provides for polynucleotide sequences comprising fragments of SEQ ID NO: 10084. In one embodiment, the polynucleotide fragment does not consist entirely of SEQ ID NO: 10033 or a known coronavirus polynucleotide sequence or a known SARS polynucleotide sequence.

The invention includes an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 10084, including the amino acid sequences of FIGS. 120A to 120F e.g. selected from the group consisting of SEQ ID NO^(S): 10085 to 10209. Preferably, the amino acid sequence comprises SEQ ID NO: 10149.

The invention also provides amino acid sequences having sequence identity to an amino acid sequence encoded by SEQ ID NO: 10084. The invention provides amino acids having sequence identity to an amino acid sequence from FIGS. 120A to 120F e.g. selected from the group consisting of SEQ ID NO^(S): 10085 to 10209.

The invention also provides fragments of amino acid sequences encoded by SEQ ID NO: 10084. The invention also provides fragments of amino acid sequences selected from the group consisting of SEQ ID NO^(S): 10085 to 10209. In one embodiment, the fragment does not consist entirely of an amino acid sequence encoded by SEQ ID NO: 10033 or an amino acid sequence of a known coronavirus or an amino acid sequence of a known SARS virus. An alignment of the matching portion of SEQ ID NO: 10033 and SEQ ID NO: 10084 is included in FIG. 121.

In one embodiment, the invention comprises an amino acid sequence comprising SEQ ID NO: 10149. An alignment of the polynucleotide sequence SEQ ID NO: 10084 to the encoded SEQ ID NO: 10149 is shown in FIG. 122 (5′3′ Frame 3). Analysis of the 5′3′ Frame 3 translation by a computer program to predict start codon methionines (NetStart 1.0) (FIG. 123) reveals SEQ ID NO^(S): 10210 to 10215.

The invention includes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10210, SEQ ID NO: 10211, SEQ ID NO: 10212, SEQ ID NO: 10213, SEQ ID NO: 10214 and SEQ ID NO: 10215. The invention includes a protein having sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10210, SEQ ID NO: 10211, SEQ ID NO: 10212, SEQ ID NO: 10213, SEQ ID NO: 10214 and SEQ ID NO: 10215. In one embodiment, the protein does not consist entirely of an amino acid sequence of a known SARS virus or of a known coronavirus.

The invention includes a fragment of a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10210, SEQ ID NO: 10211, SEQ ID NO: 10212, SEQ ID NO: 10213, SEQ ID NO: 10214 and SEQ ID NO: 10215. In one embodiment, the fragment does not consist entirely of an amino acid sequence of a known SARS virus or of a known coronavirus.

In one embodiment, the invention includes a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 10210, SEQ ID NO: 10211 and SEQ ID NO: 10212. Partial results of a BLAST of SEQ ID NO: 10210 against GenBank is included in FIG. 124. These results indicate that SEQ ID NOS: 10210, 10211 and 10212 have functional similarities to a Coronavirus RNA polymerase, particularly the RNA polymerase of murine hepatitis virus, bovine coronavirus, and avian infectious bronchitis.

In one embodiment, the invention is directed to a polypeptide comprising a first amino acid sequence selected from the group consisting of SEQ ID NO: 10210, SEQ ID NO: 10211 and SEQ ID NO: 10212 and a second amino acid sequence from the C-terminus of a coronavirus ORF1ab sequence. Preferably, the second amino acid sequence is from a bovine coronavirus. One example of this embodiment is shown below as SEQ ID NO: 10216. Amino acids 1-481 of SEQ ID NO: 10216 are the first amino acid sequence of SEQ ID NO: 10210, and amino acids 482-1152 are the second amino acid sequence of the C-terminus of a bovine coronavirus orf1ab polyprotein (Gi 26008080) (NP_(—)150073.2) (SEQ ID NO: 10217).

Accordingly, the invention includes a polypeptide comprising SEQ ID NO: 10216. The invention further includes a polypeptide comprising a first amino acid sequence of SEQ ID NO: 10210 and a second amino acid sequence of SEQ ID NO: 10217. The invention further includes a polypeptide comprising a first amino acid sequence having greater than x % identity to SEQ ID NO: 10210 and a second amino acid sequence having greater than y % identity to SEQ ID NO: 10217, wherein x is greater than or equal to 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) and wherein y is greater than or equal to 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more).

The invention also includes a polypeptide comprising a fragment of SEQ ID NO: 10210, wherein said fragment includes an epitope. Computer-predicted epitopes of SEQ ID NO: 10210, using a 17 mer window, are included in FIG. 125A (Hopp & Woods) and FIG. 125B (Kyte & Doolittle).

The amino acid sequence of SEQ ID NO: 10210 also contains two predicted glycosylation sites at amino acids 81-84 (NNTE; SEQ ID NO: 10218) and at 180-183 (NHSV; SEQ ID NO: 10219). Accordingly, the invention includes a polypeptide comprising a fragment of SEQ ID NO: 10210, wherein said fragment includes a glycosylation site. The invention further includes a polypeptide comprising a fragment of SEQ ID NO: 10210, wherein said fragment includes the Asn at position 81. Preferably, said Asn is glycosylated. The invention further includes a polypeptide comprising a fragment of SEQ ID NO: 10210, wherein said fragment includes the Asn at position 180. Preferably, said Asn is glycosylated.

In one embodiment, the invention includes a polypeptide comprising an amino acid sequence from within FIG. 120D and/or SEQ ID NO^(S): 10150 to 10160 e.g. from SEQ ID NO^(S): 10154, 10155, 10158 and 10160. Within SEQ ID NO: 10154 the following amino acid sequences starting with a Met and ending at a stop codon can be identified: SEQ ID NO^(S): 10220 to 10227.

Accordingly, the invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10220, SEQ ID NO: 10221, SEQ ID NO: 10222, SEQ ID NO: 10223, SEQ ID NO: 10224, SEQ ID NO: 10225, SEQ ED NO: 10226 and SEQ ID NO: 10227, or a fragment thereof or an amino acid sequence having sequence identity thereto.

In one embodiment, the invention includes a polypeptide comprising the amino acid sequence within FIG. 120E e.g. from SEQ ID NO^(S): 10161 to 10182, and in particular SEQ ID NOS: 10171 and 10176. Within SEQ ID NO^(S): 10171 and 10176 the following amino acid sequences starting with a Met and ending at a stop codon can be identified: SEQ ID NO: 10228 and SEQ ID NO: 10229.

Accordingly, the invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10228 and SEQ ID NO: 10229, or a fragment thereof or an amino acid sequence having sequence identity thereto.

In one embodiment, the invention includes a polypeptide comprising an amino acid sequence from FIG. 120F e.g. SEQ ID NO^(S): 10183 to 10209. Within FIG. 120F the following amino acid sequence starting with a Met and ending at a stop codon can be identified: SEQ ID NO: 10187. Accordingly, the invention includes a polypeptide comprising an amino acid sequence of SEQ ID NO: 10187, or a fragment thereof or an amino acid sequence having sequence identity thereto.

In one embodiment, the polynucleotides of the invention do not include one of the following primers, disclosed at http://content.nejm.org/cgi/reprint/NEJMoa030781v2.pdf. 5′GGGTTGGGACTATCCTAAGTGTGA3′ (SEQ ID NO: 10230) 5′TAACACACAACICCATCATCA3′ (SEQ ID NO: 10231) 5′CTAACATGCTTAGGATAATGG3′ (SEQ ID NO: 10232) 5′GCCTCTCTTGTTCTTGCTCGC3′ (SEQ ID NO: 10233) 5′CAGGTAAGCGTAAAACTCATC3′ (SEQ ID NO: 10234)

The invention also includes polynucleotide sequences which can be used as probes for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes the polynucleotide primers identified in Table 31 (SEQ ID NO^(S): 10235 to 10258), the forward primers SEQ ID NO^(S): 10259 to 10281 and the reverse primers SEQ ID NO^(S): 10282 to 10298. The invention further includes polynucleotide sequences which are complementary to any one of these primer sequences disclosed herein.

The invention provides a SARS polynucleotide sequence SEQ ID NO: 10299. All six reading frames of this sequence are included in FIG. 126 (See also FIG. 131). The constituent amino acid sequences from FIG. 126, having at least 4 amino acids, are listed as SEQ ID NOS: 10300 to 10337.

Accordingly, the invention includes a polynucleotide sequence comprising SEQ ID NO: 10299. It also provides polynucleotide sequences having sequence identity to SEQ ID NO: 10299. The invention also provides for polynucleotide sequences comprising fragments of SEQ ID NO: 10299. In one embodiment, the polynucleotide fragment does not consist entirely of a known polynucleotide sequence of a SARS virus or a known polynucleotide sequence of a coronavirus.

The invention includes an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 10299, including the amino acid sequences shown in FIG. 126, and the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 10300 to 10337. Preferably, the amino acid sequence comprises SEQ ID NO: 10316.

The invention also provides amino acid sequences having sequence identity to an amino acid sequence encoded by SEQ ID NO: 10299. The invention provides amino acid sequences having identity to an amino acid sequence selected from the group consisting of SEQ ID NO^(S): 10300 to 10337.

The invention also provides fragments of amino acid sequences encoded by SEQ ID NO: 10299. The invention also provides fragments of amino acid sequences selected from the group consisting of SEQ ID NO^(S): 10300 to 10337. In one embodiment, the fragment does not consist entirely of a known amino acid sequence of a SARS virus or a known amino acid sequence of a coronavirus.

In one embodiment, the invention comprises an amino acid sequence comprising SEQ ID NO: 10316. Encoded open reading frames within SEQ ID NO: 10316 include SEQ ID NO: 10338 and SEQ ID NO: 10339.

In one embodiment, the invention comprises an amino acid sequence comprising a sequence from within the 5′3′ Frame 1 translation of SEQ ID NO: 10299. The following encoded open reading frame is found within this translation: SEQ ID NO: 10340.

In one embodiment, the invention comprises an amino acid sequence comprising a sequence from within the 3′5′ Frame 1 translation of SEQ ID NO: 10299. An encoded open reading frame within this translation is SEQ ID NO: 10341.

In one embodiment, the invention comprises an amino acid sequence comprising a sequence from within the 3′5′ Frame 2 translation of SEQ ID NO: 10299. An encoded open reading frame within this translation is SEQ ID NO: 10342.

The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10338, SEQ ID NO: 10339, SEQ ID NO: 10340, SEQ ID NO: 10341 and SEQ ID NO: 10342. The invention includes a polypeptide having sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 10338, SEQ ID NO: 10339, SEQ ID NO: 10340, SEQ ID NO: 10341 and SEQ ID NO: 10342. The invention includes a fragment of a polypeptide comprising an amino acid sequence elected from the group consisting of SEQ ID NO: 10338, SEQ ID NO: 10339, SEQ ID NO: 10340, SEQ ID NO: 10341 and SEQ ID NO: 10342. In one embodiment, the fragment does not consist entirely of a known SARS virus amino acid sequence or of a known coronavirus amino acid sequence.

In one embodiment, SEQ ID NOS: 10338-10342 are used in fusion proteins. Accordingly, the start codon methionines may be removed. The invention comprises a amino acid sequence selected from the group consisting of SEQ ID NO: 10343, SEQ ID NO: 10344, SEQ ID NO: 10345, SEQ ID NO: 10346 and SEQ ID NO: 10347.

In one embodiment, the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10338 and SEQ ID NO: 10339. Partial BLAST results of SEQ ID NO: 10338 against GenBank are given below: >gi|133593|sp|P18457|RRPB_CVPFS   RNA-DIRECTED RNA POLYMERASE (ORF1B)  gi|93934|pir||A43489   RNA-directed RNA polymerase (EC 2.7.7.48) - porcine transmissible gastroenteritis virus (fragment)  gi|833161|emb|CAA37284.1|       polymerase  [Transmissible gastroenteritis virus]           Length = 533  Score = 131 bits (329), Expect = 3e−30  Identities = 55/89 (61%), Positives = 69/89 (77%), Gaps = 1/89 (1%). Query: 1   MLWCKDGHVETFYPKLQASQAWQPGVAMPNLYKMQRMLLEKCDLQNYGENAVIPKGIMMN 60            MLWC++ H++TFYP+LQ+++ W PG +MP LYK+QRM LE+C+L NYG    +P GI  N Sbjct: 217 MLWCENSHIKTFYPQLQSAE-WNPGYSMPTLYKIQRMCLERCNLYNYGAQVKLPDGITTN 275 Query: 61  VAKYTQLCQYLNTLTLAVPSNMRVIHFGA 89            V KYTQLCQYLNT TL VP  MRV+H GA Sbjct: 276 VVKYTQLCQYLNTTTLCVPHKMRVLHLGA 304

These results indicate that SEQ ID NO: 10338 has functional similarities to an RNA-directed RNA polymerase of porcine transmissible gastroenteritis virus.

Partial BLAST results of SEQ ID NO: 10339 against GenBank are given below: >gb|AAL57305.1|replicase [bovine coronavirus]           Length = 7094  Score = 139 bits (351), Expect = 7e−33  Identities = 64/108 (59%), Positives = 78/108 (72%) Query:    1  MSVISKVVKVTIDYAEISFMLWCKDGHVETFYPKLQASQAWQPGVAMPNLYKMQRMLLEK 60              M+ +SKVV V +D+ +  FMLWC D  V TFYP+LQA+  W+PG +MP LYK     +E+ Sbjct: 6760  LNCVSKVVNVNVDFKDFQFMLWCNDEKVMTFYPRLQAASDWKPGYSMPVLYKYLNSPMER 6819 Query:   61  CDLQNYGENAVIPKGIMMNVAKYTQLCQYLNTLTLAVPSNMRVIHFGA 108              L NYG+   +P G MMNVAKYTQLCQYLNT TLAVP NMRV+H GA Sbjct: 6820  VSLWNYGKPVTLPTGCMMNVAKYTQLCQYLNTTTLAVPVNMRVLHLGA 6867

These results indicate that SEQ ID NO: 10339 has functional similarities to a replicase of bovine coronavirus.

The SARS virus may contain polymorphism at the Glu-20 residue of SEQ ID NO: 10338. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 10338, wherein said polypeptide includes an amino acid sequence selected from the group consisting of ASQAW (SEQ ID NO: 10348) and ASRAW (SEQ ID NO: 10349). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 10338, wherein said fragment includes an amino acid sequence selected from the group consisting of SEQ ID NO: 10348 and SEQ ID NO: 10349.

The SARS virus may contain polymorphism at the Ser-80 residue of SEQ ID NO: 10338. below. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 10338, wherein said polypeptide includes an amino acid sequence selected from the group consisting of VPSNM (SEQ ID NO: 10350) and VPTNM (SEQ ID NO: 10351). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 10338, wherein said fragment includes an amino acid sequence selected from the group consisting of SEQ ID NO: 10350 and SEQ ID NO: 10351.

The invention also includes polynucleotide sequences which can be used as probes for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified in Table 32. The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in Table 32.

The invention provides a SARS polynucleotide sequence SEQ ID NO: 10505. All six reading frames of this sequence are shown in FIG. 127 (see also FIG. 132). The constituent amino acid sequences from FIG. 127, having at least 4 amino acids, are listed as SEQ ID NOS: 10506 to 10570.

The invention includes a polynucleotide sequence comprising SEQ ID NO: 10505. The invention also provides polynucleotide sequences having sequence identity to SEQ ID NO: 10505. The invention also provides for polynucleotide sequences comprising fragments of SEQ ID NO: 10505. In one embodiment, the polynucleotide fragment does not consist entirely of a known SARS virus polynucleotide sequence or of a known coronavirus polynucleotide sequence.

The invention includes an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 10505, including the amino acid sequences shown in FIG. 127, and particularly those selected from the group consisting of SEQ ID NO^(S): 10506 to 10570. Preferably, the amino acid sequence comprises SEQ ID NO: 10532 and/or SEQ ID NO: 10533.

The invention also provides amino acid sequences having sequence identity to an amino acid sequence encoded by SEQ ID NO: 10505. The invention provides amino acid sequences having sequence identity to an amino acid sequence selected from the group consisting of the sequences shown in FIG. 127, and in particular SEQ ID NO^(S): 10506 to 10570.

The invention also provides fragments of amino acid sequences encoded by SEQ ID NO: 10505. The invention also provides fragments of amino acid sequences selected from the group consisting of SEQ ID NO^(S): 10506 to 10570. In one embodiment, the fragment does not consist entirely of a known amino acid sequence of a SARS virus or a known amino acid sequence of a coronavirus.

In one embodiment, the invention includes a polypeptide comprising an amino acid sequence from the 5′3′ Frame 3 of FIG. 127. Some encoded open reading frames within this translation are: SEQ ID NO: 10533; SEQ ID NO: 10571; SEQ ID NO: 10572; SEQ ID NO: 10573; SEQ ID NO: 10574.

The invention includes a polypeptide sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10533, SEQ ID NO: 10571, SEQ ID NO: 10572, SEQ ID NO: 10573 and SEQ ID NO: 10574. The invention includes a polypeptide having sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 10533, SEQ ID NO: 10571, SEQ ID NO: 10572, SEQ ID NO: 10573 and SEQ ID NO: 10574. The invention includes a fragment of a polypeptide sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10533, SEQ ID NO: 10571, SEQ ID NO: 10572, SEQ ID NO: 10573 and SEQ ID NO: 10574.

Partial BLAST results of SEQ ID NO: 10533 against GenBank are given below: >gi|7739601|gb|AAF68926.1|AF207902_11     nucleocapsid protein [murine hepatitis virus strain ML-11]           Length = 451  Score = 147 bits (370), Expect = 3e−34  Identities = 102/252 (40%), Positives = 137/252 (54%), Gaps = 18/252 (7%) Query: 49  SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106            SWF+ +TQ  K +E +F +GQGVPI +     +Q GY+ R  RR  +  DG+ K+L PRW Sbjct: 63  SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQLLPRW 122 Query: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166            YFYYLGTGP A   YG + EG+VWVA++ A       +  R+P+++ A   +   GT LP Sbjct: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTRFAPGTVLP 182 Query: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLN 226            +GFY EGS   + AS   S        N    SS    PA          +A L+L +L Sbjct: 183 QGFYVEGSGRSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVLAKLG 242 Query: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPEQTQG 282            +        GQ +Q   VTK+SA E  +    KPRQKRT  KQ  V Q FG+RGP Q Sbjct: 243 K------DAGQPKQ---VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPNQ--- 290 Query: 283 NFGDQDLIRQGT 294            NFG  ++++ GT Sbjct: 291 NFGGSEMLKLGT 302 >gi|3132999|gb|AAC16422.1|     nucleocapsid protein [murine hepatitis virus strain 2]           Length = 451  Score = 147 bits (370), Expect = 3e−34  Identities = 102/252 (40%), Positives = 137/252 (54%), Gaps = 18/252 (7%) Query: 49  SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106            SWF+ +TQ  K +E +F +GQGVPI +     +Q GY+ R  RR  +  DG+ K+L PRW Sbjct: 63  SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQLLPRW 122 Query: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166            YFYYLGTGP A   YG + EG+VWVA++ A       +  R+P+++ A   +   GT LP Sbjct: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTKFAPGTVLP 182 Query: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLN 226            +GFY EGS   + AS   S        N    SS    PA          +A L+L +L Sbjct: 183 QGFYVEGSGKSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVLAKLG 242 Query: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPEQTQG 282            +        GQ +Q   VTK+SA E  +    KPRQKRT  KQ  V Q FG+RGP Q Sbjct: 243 K------DAGQPKQ---VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPNQ--- 290 Query: 283 NFGDQDLIRQGT 294            NFG  ++++ GT Sbjct: 291 NFGGSEMLKLGT 302 >gi|127877|sp|P03417|NCAP_CVMJH   Nucleocapsid protein  gi|174859|pir||VHIHMJ     nucleocapsid protein - murine hepatitis virus (strain JHM)  gi|58973|emb|CAA25497.1|   nucleocapsid protein [Murine hepatitis virus]           Length = 455  Score = 146 bits (369), Expect = 4e−34  Identities = 110/254 (43%), Positives = 142/254 (55%), Gaps = 22/254 (8%) Query: 49  SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106            SWF+ +TQ  K +E +F +GQGVPI        Q GY+ R  RR  +  DG+ K+L PRW Sbjct: 67  SWFSGITQFQKGKEFQFAQGQGVPIANGIPASQQKGYWYRHNRRSFKTPDGQQKQLLPRW 126 Query: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166            YFYYLGTGP A   YG + EG+VWVA++ A       I  R+P+++ A   +   GT LP Sbjct: 127 YFYYLGTGPYAGAEYGDDIEGVVWVASQQAETRTSADIVERDPSSHEAIPTRFAPGTVLP 186 Query: 167 KGFYAEGSRGGSQASSRSSSR--SRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDR 224            +GFY EGS G S  +SRS SR  SRG   N    SS    PA          +A L+L + Sbjct: 187 QGFYVEGS-GRSAPASRSGSRPQSRG-PNNRARSSSNQRQPASTVKPDMAEEIAALVLAK 244 Query: 225 LNQLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPEQT 280            L +        GQ +Q   VTK+SA E  +    KPRQKRT  KQ  V Q FG+RGP Q Sbjct: 245 LGK------DAGQPKQ---VTKQSAKEVRQKILNKPRQKRTPNKQCPVQQCFGKRGPNQ- 294 Query: 281 QGNFGDQDLIRQGT 294              NFG  ++++ GT Sbjct: 295 --NFGGPEMLKLGT 306 >gi|6625766|gb|AAF19389.1|AF201929_7           nucleocapsid protein [murine hepatitis virus strain 2]  gi|7769348|gb|AAF69338.1|AF208066_11          nucleocapsid protein [murine hepatitis virus]           Length = 451  Score = 146 bits (368), Expect = 5e−34  Identities = 102/252 (40%), Positives = 137/252 (54%), Gaps = 18/252 (7%) Query: 49  SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106            SWF+ +TQ  K +E +F +GQGVPI +     +Q GY+ R  RR  +  DG+ K+L PRW Sbjct: 63  SWFSGITQFQKGKEFQFAQGQGVPIASGIPASEQKGYWYRHNRRSFKTPDGQHKQLLPRW 122 Query: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGALNTPKDHIGTRNPNNNAATVLQLPQGTTLP 166            YFYYLGTGP A   YG + EG+VWVA++ A       +  R+P+++ A   +   GT LP Sbjct: 123 YFYYLGTGPHAGAEYGDDIEGVVWVASQQADTKTTADVVERDPSSHEAIPTRFAPGTVLP 182 Query: 167 KGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRLN 226            +GFY EGS   + AS   S        N    SS    PA          +A L+L +L Sbjct: 183 QGFYVEGSGRSAPASRSGSRSQSRGPNNRARSSSNQRQPASAVKPDMAEEIAALVLAKLG 242 Query: 227 QLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPEQTQG 282            +        GQ +Q   VTK+SA E  +    KPRQKRT  KQ  V Q FG+RGP Q Sbjct: 243 K------DAGQPKQ---VTKQSAKEVRQKILTKPRQKRTPNKQCPVQQCFGKRGPNQ--- 290 Query: 283 NFGDQDLIRQGT 294            NFG  ++++ GT Sbjct: 291 NFGGSEMLKLGT 302 >gi|21734854|gb|AAM77005.1|AF481863_7   phosphorylated nucleocapsid protein N [porcine hemagglutinating encephalomyelitis virus]           Length = 449  Score = 145 bits (366), Expect = 8e−34  Identities = 107/253 (42%), Positives = 145/253 (57%), Gaps = 18/253 (7%) Query: 49  SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106            SWF+ +TQ  K +E  F  GQGVPI       +  GY+ R  RR  +  DG  ++L PRW Sbjct: 64  SWFSGITQFQKGKEFEFAEGQGVPIAPGVPATEAKGYWYRHNRRSFKTADGNQRQLLPRW 123 Query: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGA-LNTPKDHIGTRNPNNNAATVLQLPQGTTL 165            YFYYLGTGP A   YG + +G+ WVA+  A +NTP D I  R+P+++ A   + P GT L Sbjct: 124 YFYYLGTGPHAKHQYGTDIDGVFWVASNQADINTPAD-IVDRDPSSDEAIPTRFPPGTVL 182 Query: 166 PKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRL 225            P+G+Y EGS G S  +SRS+SR+  N   S    SR NS  R ++ G    +A    D++ Sbjct: 183 PQGYYIEGS-GRSAPNSRSTSRA-PNRAPSAGSRSRANSGNRTSTPGVTPDMA----DQI 236 Query: 226 NQLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPEQTQ 281              L     GK    + Q VTK++A E  +    KPRQKR+  KQ  V Q FG+RGP Q Sbjct: 237 ASLVLAKLGK-DATKPQQVTKQTAKEVRQKILNKPRQKRSPNKQCTVQQCFGKRGPNQ-- 293 Query: 282 GNFGDQDLIRQGT 294             NFG  ++++ GT Sbjct: 294 -NFGGGEMLKLGT 305 >gi|23295765|gb|AAL80036.1|             nucleocapsid    protein    [porcine hemagglutinating encephalomyelitis virus]           Length = 449  Score = 145 bits (365), Expect = 1e−33  Identities = 107/253 (42%), Positives = 145/253 (57%), Gaps = 18/253 (7%) Query: 49  SWFTALTQHGK-EELRFPRGQGVPINTNSGPDDQIGYYRRATRR-VRGGDGKMKELSPRW 106            SWF+ +TQ  K +E  F  GQGVPI       +  GY+ R  RR  +  DG  ++L PRW Sbjct: 64  SWFSGITQFQKGKEFEFAEGQGVPIAPGVPSTEAKGYWYRHNRRSFKTADGNQRQLLPRW 123 Query: 107 YFYYLGTGPEASLPYGANKEGIVWVATEGA-LNTPKDHIGTRNPNNNAATVLQLPQGTTL 165            YFYYLGTGP A   YG + +G+ WVA+  A +NTP D I  R+P+++ A   + P GT L Sbjct: 124 YFYYLGTGPHAKDQYGTDIDGVFWVASNQADINTPAD-IVDRDPSSDEAIPTRFPPGTVL 182 Query: 166 PKGFYAEGSRGGSQASSRSSSRSRGNSRNSTPGSSRGNSPARMASGGGETALALLLLDRL 225            P+G+Y EGS G S  +SRS+SR+  N   S    SR NS  R ++ G    +A    D++ Sbjct: 183 PQGYYIEGS-GRSAPNSRSTSRA-PNRAPSAGSRSRANSGNRTSTPGVTPDMA----DQI 236 Query: 226 NQLESKVSGKGQQQQGQTVTKKSAAEASK----KPRQKRTATKQYNVTQAFGRRGPEQTQ 281              L     GK    + Q VTK++A E  +    KPRQKR+  KQ  V Q FG+RGP Q Sbjct: 237 ASLVLAKLGK-DATKPQQVTKQTAKEVRQKILNKPRQKRSPNKQCTVQQCFGKRGPNQ-- 293 Query: 282 GNFGDQDLIRQGT 294             NFG  ++++ GT Sbjct: 294 -NFGGGEMLKLGT 305 These results indicate that SEQ ID NO: 10533 has functional similarities to a coronavirus nucleocapsid protein.

In one embodiment, the invention comprises an amino acid sequence from the 5′3′ Frame 1 of FIG. 127 e.g. SEQ ID NO^(S): 10506-10514. Some encoded open reading frames within this region are SEQ ID NO^(S): 10575 to 10578.

Accordingly, the invention includes a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 10575, SEQ ID NO: 10576, SEQ ID NO: 10577 and SEQ ID NO: 10578. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to a sequence selected from the group consisting of SEQ ID NO: 10097, SEQ ID NO: 10576, SEQ ID NO: 10577 and SEQ ID NO: 10578. The invention includes a fragment of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10097, SEQ ID NO: 10576, SEQ ID NO: 10577 and SEQ ID NO: 10578.

In one embodiment, the invention includes a polypeptide comprising an amino acid sequence from the 3′5′ Frame 2 of FIG. 127 e.g. SEQ ID NO^(S): 10547-10559. An open reading frame within this region is SEQ ID NO: 10579.

The invention includes a polypeptide comprising an amino acid sequence of SEQ ID NO: 10579. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 10579. The invention includes a fragment of a polypeptide comprising an amino acid sequence of SEQ ID NO: 10579.

The invention also includes polynucleotide sequences which can be used as probes for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified in Table 33. The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified in Table 33.

The invention includes a polynucleotide sequence comprising SEQ ID NO: 11323. A polypeptide encoded by SEQ ID NO: 11323 is SEQ ID NO: 11324.

The invention includes a polypeptide comprising SEQ ID NO: 11324, sequence having sequence identity to SEQ ID NO: 11324 and fragments of SEQ ID NO: 11324. The invention includes a fragment of SEQ ID NO: 11324, wherein said polypeptide fragment begins with a Methionine.

Accordingly, the invention includes a polynucleotide sequence comprising SEQ ID NO: 11323. It also provides polynucleotide sequences having sequence identity to SEQ ID NO: 11323. The invention also provides for polynucleotide sequences comprising fragments of SEQ ID NO: 11323. In one embodiment, the polynucleotide fragment does not consist entirely of a known SARS polynucleotide sequence or a known coronavirus polynucleotide sequence.

The invention includes an amino acid sequence encoded by the polynucleotide sequence SEQ ID NO: 11323, including the amino acid sequence of SEQ ID NO: 11324.

The invention also provides amino acid sequences having sequence identity to an amino acid sequence encoded by SEQ ID NO: 11323. The invention provides amino acid sequences having sequence identity to SEQ ID NO: 11324.

The invention provides fragments of amino acid sequences encoded by SEQ ID NO: 11323. The invention also provides fragments of amino acid sequences of SEQ ID NO: 11324. In one embodiment, the fragment does not consist entirely of a known SARS amino acid sequence or a known coronavirus amino acid sequence.

The invention also includes polynucleotide sequences which can be used as probes for diagnostic reagents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample. The invention includes a polynucleotide sequence comprising one or more of the primer sequences identified as SEQ ID NO_(S): 11325-11440 (left part) and SEQ ID NO^(S): 11441-11551 (right part). The invention further includes polynucleotide sequence comprising the complement of one or more of the primer sequences identified as SEQ ID NO^(S): 11325-11551.

The invention includes a polypeptide comprising SEQ ID NO: 11552. The SARS virus contains polymorphism at the Isoleucine residue Ile-324. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11552, wherein said polypeptide includes an amino acid sequence selected from the group consisting of YSYAI (SEQ ID NO: 11553), SYAIH (SEQ ID NO: 11554), YAIHH (SEQ ID NO: 11555), IHHDK (SEQ ID NO: 11556), SYAI (SEQ ID NO: 11557), YAIH (SEQ ID NO: 11558), AIHH (SEQ ID NO: 11559), IHHD (SEQ ID NO: 11560), YAI, AIH, and IHH. The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11552, wherein said fragment includes an amino acid sequence selected from the group consisting of YSYAI (SEQ ID NO: 11553), SYAIH (SEQ ID NO: 11554), YAIHH (SEQ ID NO: 11555), IHHDK (SEQ ID NO: 11556), SYAI (SEQ ID NO: 11557), YAIH (SEQ ID NO: 11558), AIHH (SEQ ID NO: 11559), IHHD (SEQ ID NO: 11560), YAI, AIH, and IHH.

The invention includes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11561 and SEQ ID NO: 11562. The invention includes a fragment of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 11561 and SEQ ID NO: 11562.

The invention includes a diagnostic kit comprising a polypeptide comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 11561 and 11562. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding a polypeptide comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 11561 and 11562. The invention includes an immunogenic composition comprising a polypeptide comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 11561 and 11562. The invention includes an antibody which recognizes a polypeptide comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NO^(S): 11561 and 11562.

The invention includes a polynucleotide sequence SEQ ID NO: 11563 or a fragment thereof or a sequence having sequence identity thereto. Polypeptide sequences which can be translated from SEQ ID NO: 11563 are shown in FIG. 128. The constituent amino acid sequences from FIG. 128, having at least 4 amino acids, are listed as SEQ ID NO^(S): 11564 to 11617.

The invention includes a polypeptide sequence selected from the group consisting of the sequences of FIG. 128, or a fragment thereof or a sequence having sequence identity thereto e.g. SEQ ID NO^(S): 11563 to 11617.

A polypeptide sequence within SEQ ID NO: 11600 is SEQ ID NO: 11618. The invention includes a polypeptide comprising SEQ ID NO: 11618, or a fragment thereof or a sequence having sequence identity thereto.

A polypeptide sequence within SEQ ID NO: 11602 is SEQ ID NO: 11641. The invention includes a polypeptide comprising SEQ ID NO: 11641, or a fragment thereof or a sequence having sequence identity thereto.

A polypeptide sequence within SEQ ID NO: 11609 is SEQ ID NO: 11619.

The invention includes a polynucleotide encoding (i) an amino acid sequence selected from the group consisting of: (1) the amino acid sequences of FIG. 128, and in particular SEQ ID NO^(S): 11564-11617; (2) SEQ ID NO: 11618; and (3) SEQ ID NO: 11619, or (ii) a fragment thereof. The invention includes a diagnostic kit comprising a one or more of these proteins. The invention includes a diagnostic kit comprising a polynucleotide sequence encoding one or more of these polypeptide sequences. The invention includes an antibody which recognizes one or more of the polypeptide sequences.

The SARS virus may contain polymorphism at isoleucine residue Ile-326 in SEQ ID NO: 11620 (Chi-PEP3). The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11620, wherein said polypeptide includes an amino acid sequence selected from the group consisting of YAIHH (SEQ ID NO: 11621) and YATHH (SEQ ID NO: 11622). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11620, wherein said fragment includes an amino acid sequence selected from the group consisting of YAIHH (SEQ ID NO: 11621) and YATHH (SEQ ID NO: 11622).

The SARS virus may contain polymorphism at glutamine residue Gln-830 in SEQ ID NO: 11620. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11620, wherein said polypeptide includes an amino acid sequence selected from the group consisting of ASQAW (SEQ ID NO: 11623) and ASRAW (SEQ ID NO: 11624). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11620, wherein said fragment includes an amino acid sequence selected from the group consisting of ASQAW (SEQ ID NO: 11623) and ASRAW (SEQ ID NO: 11624).

The SARS virus may contain polymorphism at aspartic acid residue Asp-935 in SEQ ID NO: 11620. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11620, wherein said polypeptide includes an amino acid sequence selected from the group consisting of DADST (SEQ ID NO: 11625) and DAYST (SEQ ID NO: 11626). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11620, wherein said fragment includes an amino acid sequence selected from the group consisting of DADST (SEQ ID NO: 11625) and DAYST (SEQ ID NO: 11626).

The SARS virus may contain polymorphism at serine residue Ser-577 in SEQ ID NO: 11627 (Chi-PEP4). The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11627, wherein said polypeptide includes an amino acid sequence selected from the group consisting of PCSFG (SEQ ID NO: 11628) and PCAFG (SEQ ID NO: 11629). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11627, wherein said fragment includes an amino acid sequence selected from the group consisting of PCSFG (SEQ ID NO: 11628) and PCAFG (SEQ ID NO: 11629).

The SARS virus may contain polymorphism at valine residue Val-68 in SEQ ID NO: 11630 (Chi-PEP8). The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11630, wherein said polypeptide includes an amino acid sequence selected from the group consisting of LAVVY (SEQ ID NO: 11631) and LAAVY (SEQ ID NO: 11632). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11630, wherein said fragment includes an amino acid sequence selected from the group consisting of LAVVY (SEQ ID NO: 11631) and LAAVY (SEQ ID NO: 11632).

The SARS virus may contain polymorphism at isoleucine residue Ile-50 in SEQ ID NO: 11633 (Chi-PEP13). The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11633, wherein said polypeptide includes an amino acid sequence selected from the group consisting of NNIAS (SEQ ID NO: 11634) and NNTAS (SEQ ID NO: 11635). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11633, wherein said fragment includes an amino acid sequence selected from the group consisting of NNIAS (SEQ ID NO: 11634) and NNTAS (SEQ ID NO: 11635).

The SARS virus may contain a polymorphism at Serine residue Ser-943 in SEQ ID NO: 11636. The invention includes a polypeptide comprising an amino acid sequence having sequence identity to SEQ ID NO: 11636, wherein said polypeptide includes an amino acid sequence selected from the group consisting of AVSAC (SEQ ID NO: 11637) and AVGAC (SEQ ID NO: 11638). The invention includes a fragment of a polypeptide comprising SEQ ID NO: 11636, wherein said fragment includes an amino acid seuence selected from the group consisting of AVSAC (SEQ ID NO: 11637) and AVGAC (SEQ ID NO: 11638).

The invention includes a polynucleotide SEQ ID NO: 11639, or a fragment thereof or a sequence having sequence identity thereto. The invention includes a polypeptide encoded by the polynucleotide sequence set forth in SEQ ID NO: 11639, or a fragment thereof or a polypeptide sequence having sequence identity thereto.

The invention includes a polynucleotide set forth in SEQ ID NO: 11640, or a fragment thereof or a sequence having sequence identity thereto. The invention includes a polypeptide encoded by the polynucleotide sequence set forth in SEQ ID NO: 11640, or a fragment thereof or a polypeptide sequence having sequence identity thereto.

The invention includes each of the polynucleotides identified above. The invention includes each of the polynucleotides set forth in the sequence listing. The invention further includes polynucleotides having sequence identity to each of the polynucleotides identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

The invention includes polynucleotide sequences comprising fragments of each of the polynucleotide sequences identified above. The fragments should comprise at least n consecutive polynucleotides from a particular SEQ ID NO:, and, depending on the sequence, n is 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more).

The invention includes each of the amino acid sequences encoded by each of the polynucleotide sequences identified above. The invention includes each of the amino acid sequences encoded by each of the polynucleotide sequences set forth in the sequence listing. The invention further includes amino acid sequences having sequence identity to the amino acid sequences encoded by each of the polynucleotide sequences identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). The invention further includes fragments of amino acid sequences encoded by each of the polynucleotide sequences identified above. The fragments should comprise at least n consecutive amino acids from a particular SEQ ID NO:, and, depending on the sequence, n is 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more).

The invention includes each of the amino acid sequences identified above. The invention includes each of the amino acid sequence set forth in the sequence listing. The invention further includes amino acid sequences having sequence identity to each of the amino acid sequences identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

The invention further includes fragments of the amino acid sequences identified above. The fragments should comprise at least n consecutive amino acids from a particular SEQ ID NO:, and, depending on the sequence, n is 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more).

The invention includes polynucleotides encoding each of the amino acid sequences identified above. The invention includes polynucleotides encoding each of the amino acid sequences set forth in the sequence listing. The invention further includes polynucleotides having sequence identity with each of the polynucleotides encoding each of the amino acid sequences identified above. The degree of sequence identity is preferably greater than 50% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).

The invention further includes fragments of polynucleotides encoding each of the amino acid sequences identified above. The fragments should comprise at least n consecutive polynucleotides from a particular SEQ ID NO:, and, depending on the sequence, n is 7 or more (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more).

As described in more detail below, polynucleotides for use as primers and/or as probes may contain at least 4 or 8 contiguous nucleotides from a polynucleotide sequence of the invention e.g. at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides and up to about 50, 75, 100, 200 contiguous nucleotides or more. While 6-8 nucleotides may be a workable length, sequences of 10-12 nucleotides are preferred, and about 13, 14, 15, 16, 17, 18, 19, 20, or 21 or more nucleotides or more appears optimal for hybridisation.

In one embodiment, the invention is directed to polynucleotides and amino acid sequences that do not consist entirely of a known SARS virus polynucleotide or amino acid sequence or of a known coronavirus polynucleotide or amino acid sequence. In one embodiment, the polynucleotides and amino acid sequences of the invention do not consist entirely of the sequence SEQ ID NO: 1. In another embodiment, the polynucleotides and amino acid sequences of the invention do not consist entirely of the sequence SEQ ID NO: 2. SEQ ID NO: 9967 is a SARS genome sequence of the Frankfurt (FRA) isolate (GenBank: AY310120). Compared to SEQ ID NO: 1, it differs at nucleotides 2546, 2590, 11437, 18954, 19073, 20585, 20899, 23209, 24922, 26589 & 28257; compared to SEQ ID NO:2, it differs at nucleotides 2560, 7922, 11451, 16625, 18968 & 19067. Further genome sequences have become available from GenBank, since this application was originally filed, under accession numbers including AY559097, AY559096, AY559095, AY559094, AY559093, AY559092, AY559091, AY559090, AY559089, AY559088, AY559087, AY559086, AY559085, AY559084, AY559083, AY559082, AY559081, AY274119, AY323977, AY291315, AY502932, AY502931, AY502930, AY502929, AY502928, AY502927, AY502926, AY502925, AY502924, AY502923, AY291451, AY390556, AY395003, AY395002, AY395001, AY395000, AY394999, AY394998, AY394997, AY394996, AY394995, AY394994, AY394993, AY394992, AY394991, AY394990, AY394989, AY394987, AY394986, AY394985, AY394983, AY394979, AY394978, AY508724, AY394850, AY463059, AY463060, AY313906, AY310120, AY461660, AY485278, AY485277, AY345988, AY345987, AY345986, AY282752, AY357076, AY357075, AY350750, AY304495, AY304488, AY304486, AY427439, AY283798, AY278491, AY278489, AY362699, AY362698, AY283797, AY283796, AY283795, AY283794, AY278741, AY351680, AP006561, AP006560, AP006559, AP006558, AP006557, AY278554, AY348314, AY338175, AY338174, AY321118, AY279354, AY278490, AY278487, AY297028, AY278488, and ND_(—)004718.

In another embodiment, the invention is directed to polynucleotides that encode proteins which are not immunologically cross reactive with a protein of a mouse hepatitis virus, a bovine coronavirus or an avian infectious bronchitis virus. In another embodiment, the invention is directed to proteins which are not immunologically cross reactive with a protein of a mouse hepatitis virus, a bovine coronavirus or an avian infectious bronchitis virus.

Each of the polynucleotides identified above may be used to encode a portion of a fusion protein. Accordingly, the invention compries one or more of the polynucleotides identified above wherein the polynucleotides encoding for the start codon are removed. The invention further comprises one or more of the amino acids identified above wherein the starting methionine is removed.

Any of the polynucleotide or amino acid sequences discussed above may be used in vaccines for the treatment or prevention of SARS virus infection, including as a SARS viral antigen. Additionally, any of the polynucleotides or amino acid sequences discussed above may be used as diagnostic reagents, or in kits (comprising such reagents) or in methods used to diagnose or identify the presence or absence of a SARS virus in a biological sample.

SARS viral antigens of the invention may include a polypeptide with 99%, 95%, 90%, 85%, or 80% homology to one or more of the group consisting of the following proteins: nonstructural protein 2 (NS2); hemagglutinin-esterase glycoprotein (HE) (also referred to as E3), spike glycoprotein (S) (also referred to as E2), nonstructural region 4 (NS4), envelope (small membrane) protein (E) (also referred to as sM), membrane glycoprotein (M) (also referred to as E1), nucleocapsid phosphoprotein (N) or RNA dependent RNA polymerase (pol).

A detailed discussion of Coroavirus biology can be found in Fields Virology (2nd ed), Fields et al. (eds.), B.N. Raven Press, New York, N.Y., Chapter 35.

Another example of a SARS virus isolate is set forth in Example 1 below. The invention includes each of the polypeptide and polynucleotide sequences identified in Example 1. In addition, the invention includes vaccine formulations comprising one or more of the polypeptide or polynucleotide sequences identified in Example 1. The invention includes diagnostic regaents, kits (comprising such reagents) and methods which can be used to diagnose or identify the presence or absence of a SARS virus in a biological sample using one or more of the polypeptide or polynucleotide sequences identified in Example 1. The invention includes methods for the treatment or prevention of SARS virus infection utilizing small molecule viral inhibitors and combinations of small molecule viral inhibitors and kits for the treatment of SARS. The small molecule inhibitors may specifically target one or more of the polypeptides or polynucleotides identified in Example 1.

Further discussion of terms used in the application follows below.

“Respiratory Virus” as used herein refers to a virus capable of infecting the human respiratory tract. Respiratory Viral Antigens suitable for use in the invention include Severe Acute Respiratory Syndrome virus, coronavirus, influenza virus, human rhinovirus (HRV), parainfluenza virus (PWV), respiratory syncytial virus (RSV), adenovirus, metapneumovirus, and rhinovirus.

The terms “polypeptide”, “protein” and “amino acid sequence” as used herein generally refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, mulimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. Minimum fragments of polypeptides useful in the invention can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or even 15 amino acids. Typically, polypeptides useful in this invention can have a maximum length suitable for the intended application. Generally, the maximum length is not critical and can easily be selected by one skilled in the art.

Polypeptides of the invention can be prepared in many ways e.g. by chemical synthesis (at least in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself (e.g. after viral culture, or direct from patients), from a cell line source etc. A preferred method for production of peptides <40 amino acids long involves in vitro chemical synthesis (Bodanszky (1993) Principles of Peptide Synthesis (ISBN: 0387564314); Fields et al. (1997) Methods in Enzymology 289: Solid-Phase Peptide Synthesis. ISBN: 0121821900). Solid-phase peptide synthesis is particularly preferred, such as methods based on t-Boc or Fmoc (Chan & White (2000) Fmoc Solid Phase Peptide Synthesis ISBN: 0199637245) chemistry. Enzymatic synthesis (Kullmann (1987) Enzymatic Peptide Synthesis. ISBN: 0849368413) may also be used in part or in full. As an alternative to chemical synthesis, biological synthesis may be used e.g. the polypeptides may be produced by translation. This may be carried out in vitro or in vivo. Biological methods are in general restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e.g. of aminoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non natural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) (Ibba (1996) Biotechnol Genet Eng Rev 13:197-216.). Where D-amino acids are included, however, it is preferred to use chemical synthesis. Polypeptides of the invention may have covalent modifications at the C-terminus and/or N-terminus, particularly where they are for in vivo administration e.g by attachment of acetyl or carboxamide, as in the Fuzeon™ product.

Reference to polypeptides and the like also includes derivatives of the amino acid sequences of the invention. Such derivatives can include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of.hosts which produce the proteins or errors due to PCR amplification. Furthermore, modifications may be made that have one or more of the following effects: reducing toxicity; facilitating cell processing (e.g., secretion, antigen presentation, etc.); and facilitating presentation to B-cells and/or T-cells.

“Fragment” or “Portion” as used herein refers to a polypeptide consisting of only a part of the intact full-length polypeptide sequence and structure as found in nature. For instance, a fragment can include a C-terminal deletion and/or an N-terminal deletion of a protein.

A “recombinant” protein is a protein which has been prepared by recombinant DNA techniques as described herein. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expressed the foreign gene to produce the protein under expression conditions.

The term “polynucleotide”, as known in the art, generally refers to a nucleic acid molecule. A “polynucleotide” can include both double- and single-stranded sequences and refers to, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic RNA and DNA sequences from viral (e.g. RNA and DNA viruses and retroviruses) or prokaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA, and includes modifications such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the nucleic acid molecule encodes a therapeutic or antigenic protein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the antigens. Modifications of polynucleotides may have any number of effects including, for example, facilitating expression of the polypeptide product in a host cell.

Polynucleotides of the invention may be prepared in many ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases (e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides (e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.

A polynucleotide can encode a biologically active (e.g., immunogenic or therapeutic) protein or polypeptide. Depending on the nature of the polypeptide encoded by the polynucleotide, a polynucleotide can include as little as 10 nucleotides, e.g., where the polynucleotide encodes an antigen.

By “isolated” is meant, when referring to a polynucleotide or a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose. The polynucleotides and polypeptides of the invention are preferably isolated polynucleotides and isolated polypeptides.

“Antibody” as known in the art includes one or more biological moieties that, through chemical or physical means, can bind to or associate with an epitope of a polypeptide of interest. The antibodies of the invention include antibodies which specifically bind to a SARS viral antigen. The term “antibody” includes antibodies obtained from both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349: 293-299; and U.S. Pat. No. 4,816,567; F(ab′)₂ and F(ab) fragments; F_(v) molecules (non-covalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5897-5883); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, for example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties of the parent antibody molecule. The term “antibody” further includes antibodies obtained through non-conventional processes, such as phage display.

As used herein, the term “monoclonal antibody” refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. Thus, the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas. See, e.g., Cote, et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p 77.

An “immunogenic composition” as used herein refers to a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest. The immunogenic composition can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal or any other parenteral, mucosal or transdermal (e.g., intra-rectally or intra-vaginally) route of administration.

The term “derived from” is used to identify the source of molecule (e.g., a molecule can be derived from a polynucleotide, polypeptide, an immortalized cell line can be derived from any tissue, etc.). A first polynucleotide is “derived from” a second polynucleotide if it has the same or substantially the same basepair sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above. Thus, a first polynucleotide sequence is “derived from” a second sequence if it has (i) the same or substantially the same sequence as the second sequence or (ii) displays sequence identity to polypeptides of that sequence.

A first polypeptide is “derived from” a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above. Thus, a polypeptide (protein) is “derived from” a particular SARS virus if it is (i) encoded by an open reading frame of a polynucleotide of that SARS virus, or (ii) displays sequence identity, as described above, to polypeptides of that SARS virus.

Both polynucleotide and polypeptide molecules can be physically derived from a SARS virus or produced recombinantly or synthetically, for example, based on known sequences.

A cultured cell or cell line is “derived from” another cell, cells or tissue if it is originally obtained from existing cells or tissue. Non-limiting examples of tissue that cells may be derived from include skin, retina, liver, kidney, heart, brain, muscle, intestinal, ovary, breast, prostate, cancerous tissue, tissue infected with one or more pathogens (e.g., viruses, bacteria etc.) and the like. The cells described herein may also be derived from other cells including, but not limited to, primary cultures, existing immortalized cells line and/or other isolated cells.

An “antigen” refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” Normally, an epitope will include between about 3-15, generally about 5-15 amino acids. A B-cell epitope is normally about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term “antigen” denotes both subunit antigens, (i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature), as well as, killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes as well as tumor antigens, including extracellular domains of cell surface receptors and intracellular portions that may contain T-cell epitopes. Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein. Similarly, an oligonucleotide or polynucleotide that expresses an antigen or antigenic determinant in vivo, such as in gene therapy and DNA immunization applications, is also included in the definition of antigen herein.

An “immunological response” to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest. For purposes of the present invention, a “humoral immune response” refers to an immune response mediated by antibody molecules, including secretory (IgA) or IgG molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTL”s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells. In addition, a chemokine response may be induced by various white blood or endothelial cells in response to an administered antigen.

II. Vaccine Formulations

The invention relates to vaccine formulations for the treatment or prevention of Severe Acute Respiratory Syndrome (SARS). Vaccine formulations of the invention include an inactivated (or killed) SARS virus, an attenuated SARS virus, a split SARS virus preparation and a recombinant or purified subunit formulation of one or more SARS viral antigens. The invention includes polypeptides and polynucleotides encoding for SARS viral antigens and immunogenic fragments thereof. Expression and delivery of the polynucleotides of the invention may be facilitated via viral vectors and/or viral particles, including Virus Like Particles (VLPs).

A. Inactivated (or Killed) SARS Vaccines

The invention includes a composition comprising an inactivated (or killed) SARS virus and methods for the production thereof. Inactivated SARS viral compositions can be used as prophylactic or therapeutic SARS virus vaccine. Preferably the inactivated SARS virus vaccine composition comprises an amount of inactivated SARS virus which, before inactivation, is equivalent to a virus titer of from about 4 to 7 logs plaque forming units (PFU) or 4 to 7 logs tissue culture infectious dose 50 (TCID₅₀) per milliliter. More preferably, before inactivation the virus titer is from 4 to 11, 7 to 11 or 9 to 11 PFU or TCID₅₀. Still more preferably the inactivated SARS virus vaccine composition comprises an amount of inactivated SARS virus which, before inactivation, is equivalent to a virus titer of from about 5 to 9 PFU or 5 to 9 TCID₅₀ per milliliter. In one embodiment, the PFU or TCID₅₀ of the cultured SARS virus at harvest is 6 to 8, more preferably about 7.5 PFU or TCID₅₀ per milliliter. Upon concentration of the viral harvest, the PFU or TCID₅₀ is preferably 8 to 11, still more preferably about 9 PFU or TCID₅₀ per milliliter. The vaccine composition comprises a sufficient amount of the SARS virus antigen to produce an immunological response in a primate.

Methods of inactivating or killing viruses are known in the art to destroy the ability of the viruses to infect mammalian cells. Such methods include both chemical or physical means. Chemical means for inactivating a SARS virus include treatment of the virus with an effective amount of one or more of the following agents: detergents, formaldehyde, formalin, β-propiolactone, or UV light. Additional chemical means for inactivation include treatment with methylene blue, psoralen, carboxyfullerene (C60) or a combination of any thereof. Other methods of viral inactivation are known in the art, such as for example binary ethylamine, acetyl ethyleneimine, or gamma irradiation.

For example formaldehyde may be used at concentrations such as 0.1 to 0.02%, preferably at 0.02 to 0.1%, and still more preferably at 0.04 to 0.05%. The inactivating agent is added to virus containing culture supernatants, prior to or after harvesting said culture supernatants from vessels used for virus propagation, either with or without a step of cell disruption for release of cell-associated virus prior to harvesting. Further, the inactivating agent may be added after said culture supernatants have been stored frozen and thawed, or after one or more steps of purification to remove cell contaminants. Preferably, however, formaldehyde is added after removal of cells and cellular debris or after one or more purification steps. After addition of formaldehyde, the virus containing mixture is transferred into an incubation vessel and incubated at refrigeration temperatures (e.g. +2 to 8° C.) or alternatively at elevated temperatures, such as ambient temperatures between approximately 20 and 30° C. or at 33° C. to 37° C. for a period of 12 hours to 7 days, whereby the temperature chosen should be adjusted to the duration of incubation. Prefered conditions are e.g. +2-8° C. for 3-7 days (prefered are 3-7days), ambient temperatures and incubation for 16 hours to 3 days (prefered 24-48 hours), or 35-37° C. for 12-36 hours. If it is desirable to remove excess formalin, sodium thiosulfate or sodium metabisulfite at equimolar or 1.5-fold molar concentration (relative to formaldehyde) may be added after completing the inactivation process.

For example, β-propiolactone may be used at concentrations such as 0.01 to 0.5%, preferably at 0.5% to 0.2%, and still more preferably at 0.025 to 0.1%. The inactivating agent is added to virus containing culture supernatants (virus material) prior to or after harvesting said culture supernatants from vessels used for virus propagation, either with or without a step of cell disruption for release of cell-associated virus prior to harvesting. Further, the inactivating agent may be added after said culture supernatants have been stored frozen and thawed, or after one or more steps of purification to remove cell contaminants. β-propiolactone is added to the virus material, with the adverse shift in pH to acidity being controlled with sodium hydroxide (e.g., 1 N NaOH), a Tris-buffer or sodium bicarbonate solution. After transfering the mixture to another inactivation vessel, the combined inactivating agent-virus materials are incubated at temperatures from 4° C. to 37° C., for incubation times of preferably 24 to 72 hours.

Another inactivant which may be used is binary ethyleneimine (BEI). Equal volumes of a 0.2 molar bromoethylamine hydrobromide solution and a 0.4 molar sodium hydroxide solution are mixed and incubated at about 37° C. for 60 minutes. The resulting cyclized inactivant is binary ethyleneimine, which is added to the virus materials at 0.5 to 4 percent, and preferably at 1 to 3 percent, volume to volume. The inactivating virus materials are held from about 4° C. to 37° C. for 24 to 72 hours with periodic agitation. At the end of this incubation 20 ml. of a sterile 1 molar sodium thiosulfate solution was added to insure neutralization of the BEI.

In one embodiment, the invention includes an inactivating method is designed to maximize exposure of the virus to the inactivating agent and to minimize long-term exposure of the temperature sensitive SARS virus particles to elevated temperatures. The invention includes an inactivation method comprising exposing the virus to the inactivation agent (such as BPL) for 12 to 24 hours at refrigeration temperatures followed by hydrolysis of any residual inactivating agent by elevating the temperature for only 3 hours. Preferably, the refrigeration temperatures are between 0 and 8° C., more preferably around 4° C. Preferably, the elevated temperature is between 33 and 41° C., more preferably around 37° C. As assessed by a test for residual infectious virus using 10 ml aliquots of the inactivated preparation, the method is able to inactivate SARS-CoV in raw cell culture harvests below a theoretical limit of 0.03 infectious units/ml.

Diluted and undiluted samples of the inactivated virus materials are added to susceptible cell (tissue) culture (e.g., VERO) to detect any non-inactivated virus. The cultured cells are passaged multiple times and examined for the presence of SARS virus based on any of a variety of methods, such as, for example, cytopathic effect (CPE) and antigen detection (e.g., via fluoroscent antibody conjugates specific for SARS virus). Such tests allow determination of complete virus inactivation.

Prior to inactivation, the SARS virus will be cultured in a mammalian cell culture. The cell culture may be adherently growing cells or cells growing in suspension. Preferably the cells are of mammalian origin, but may also be derived from avian (e.g., hens' cells such as hens' embryo cells (CEF cells)), amphibian, reptile, insect, or fish sources. Mammalian sources of cells include, but are not limited to, human or non-human primate (e.g., MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HeLa cells, human diploid cells, fetal rhesus lung cells (e.g. ATCC CL-160), human embryonic kidney cells (293 cells, typically transformed by sheared adenovirus type 5 DNA), VERO cells (e.g., from monkey kidneys), horse, cow (e.g., MDBK cells), sheep, dog (e.g., MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 as described in WO 97/37001), cat, and rodent (e.g., hamster cells such as BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and may be obtained from a wide variety of developmental stages, including for example, adult, neonatal, fetal, and embryo.

In certain embodiments the cells are immortalized (e.g., PERC.6 cells are described, for example, in WO 01/38362 and WO 02/40665, incorporated by reference herein in their entireties, as well as deposited under ECACC deposit number 96022940), or any other cell type immortalized using the techniques described herein.

In preferred embodiments, mammalian cells are utilized, and may be selected from and/or derived from one or more of the following non-limiting cell types: fibroblast cells (e.g., dermal, lung), endothelial cells (e.g., aortic, coronary, pulmonary, vascular, dermal microvascular, umbilical), hepatocytes, keratinocytes, immune cells (e.g., T cell, B cell, macrophage, NK, dendritic), mammary cells (e.g., epithelial), smooth muscle cells (e.g., vascular, aortic, coronary, arterial, uterine, bronchial, cervical, retinal pericytes), melanocytes, neural cells (e.g., astrocytes), prostate cells (e.g., epithelial, smooth muscle), renal cells (e.g., epithelial, mesangial, proximal tubule), skeletal cells (e.g., chondrocyte, osteoclast, osteoblast), muscle cells (e.g., myoblast, skeletal, smooth, bronchial), liver cells, retinoblasts, and stromal cells. WO 97/37000 and WO 97/37001, incorporated by reference herein in their entireties, describe production of animal cells and cell lines that capable of growth in suspension and in serum free media and are useful in the production and replication of viruses.

Preferably, the SARS viruses of the invention are grown on VERO cells or fetal rhesus kidney cells.

Culture conditions for the above cell types are well-described in a variety of publications, or alternatively culture medium, supplements, and conditions may be purchased commercially, such as for example, as described in the catalog and additional literature of Cambrex Bioproducts (East Rutherford, N.J.).

In certain embodiments, the host cells used in the methods described herein are cultured in serum free and/or protein free media. A medium is referred to as a serum-free medium in the context of the present invention in which there are no additives from serum of human or animal origin. Protein-free is understood to mean cultures in which multiplication of the cells occurs with exclusion of proteins, growth factors, other protein additives and non-serum proteins. The cells growing in such cultures naturally contain proteins themselves.

Known serum-free media include Iscove's medium, Ultra-CHO medium (BioWhittaker) or EX-CELL (JRH Bioscience). Ordinary serum-containing media include Eagle's Basal Medium (BME) or Minimum Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium (DMEM or EDM), which are ordinarily used with up to 10% fetal calf serum or similar additives. Optionally, Minimum Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium (DMEM or EDM) may be used without any serum containing supplement. Protein-free media like PF-CHO (JHR Bioscience), chemically-defined media like ProCHO 4CDM (BioWhittaker) or SMIF 7 (Gibco/BRL Life Technologies) and mitogenic peptides like Primactone, Pepticase or HyPep™ (all from Quest International) or lactalbumin hydrolyzate (Gibco and other manufacturers) are also adequately known in the prior art. The media additives based on plant hydrolyzates have the special advantage that contamination with viruses, mycoplasma or unknown infectious agents can be ruled out.

The cell culture conditions to be used for the desired application (temperature, cell density, pH value, etc.) are variable over a very wide range owing to the suitability of the cell line employed according to the invention and can be adapted to the requirements of the SARS virus.

The method for propagating the SARS virus in cultured cells (e.g., mammalian cells) includes the steps of inoculating the cultured cells with SARS virus, cultivating the infected cells for a desired time period for virus propagation, such as for example as determined by SARS virus titer or SARS virus antigen expression (e.g., between 24 and 168 hours after inoculation) and collecting the propagated virus. The cultured cells are inoculated with a SARS virus (measured by PFU or TCID₅₀) to cell ratio of 1:10000 to 1:10. A lower range of ratios may also be used e.g. 1:500 to 1:1, preferably 1:100 to 1:5, more preferably 1:50 to 1:10. The SARS virus is added to a suspension of the cells or is applied to a monolayer of the cells, and the virus is absorbed on the cells for at least 60 minutes but usually less than 300 minutes, preferably between 90 and 240 minutes at 25° C to 40° C., more preferably 28° C. to 37° C., still more preferably at about 33° C. The infected cell culture (e.g., monolayers) may be treated either by freeze-thawing or by enzymatic action to increase the viral content of the harvested culture supernatants. The harvested fluids are then either inactivated or stored frozen.

A comparison of SARS infected Vero cells grown with and without fetal calf serum (“FCS”) is shown in FIG. 26A. Briefly, Vero cells were split the day before infection and cultivated in T175 flasks. Infection of 90% confluent Vero cell monolayers the following day was performed with a SARS-CoV seed stock (strain FRA, passage 4, Accession number AY310120), with or without 3% FCS (FIG. 26A). The addition of FCS to the cell media showed little impact on virus yield.

Cultured cells may be infected at a multiplicity of infection (“m.o.i.”) of about 0.0001 to 10, preferably 0.002 to 5, more preferably to 0.001 to 2. Still more preferably, the cells are infected at a m.o.i of about 0.01. A comparison of viral yield at varying m.o.i. levels is shown in FIG. 26B.

Infected cells may be harvested 30 to 60 hours post infection. Preferably, the cells are harvested 34-48 hours post infection. Still more preferably, the cells are harvested 38 to 40 hours post infection. See FIG. 26C.

Methods of purification of inactivated virus are known in the art and may include one or more of, for instance gradient centrifugation, ultracentrifugation, continuous-flow ultracentrifugation and chromatography, such as ion exchange chromatography, size exclusion chromatography, and liquid affinity chromatography. Additional method of purification include ultrafiltration and dialfiltration. See J P Gregersen “Herstellung von Virussimpfstoffen aus Zellkulturen” Chapter 4.2 in Pharmazeutische Biotecnology (eds. O. Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000. See also, O'Neil et al., “Virus Harvesting and Affinity Based Liquid Chromatography. A Method for Virus Concentration and Purification”, Biotechnology (1993) 11:173-177; Prior et al., “Process Development for Manufacture of Inactivated HIV-1”, Pharmaceutical Technology (1995) 30-52; and Majhdi et al., “Isolation and Characterization of a Coronavirus from Elk Calves with diarrhea” Journal of Clinical Microbiology (1995) 35(11): 2937-2942.

Other examples of purification methods suitable for use in the invention include polyethylene glycol or ammonium sulfate precipitation (see Trepanier et al., “Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration” Journal of Virological Methods (1981) 3(4):201-211; Hagen et al., “Optimization of Poly(ethylene glycol) Precipitation of Hepatitis Virus Used to prepare VAQTA, a Highly Purified Inactivated Vaccine” Biotechnology Progress (1996) 12:406-412; and Carlsson et al., “Purification of Infectious Pancreatic Necrosis Virus by Anion Exchange Chromatography Increases the Specific Infectivity” Journal of Virological Methods (1994) 47:27-36) as well as ultrafiltration and microfiltration (see Pay et al., Developments in Biological Standardization (1985) 60:171-174; Tsurumi et al., “Structure and filtration performances of improved cuprammonium regenerated cellulose hollow fibre (improved BMM hollow fibre) for virus removal” Polymer Journal (1990) 22(12):1085-1100; and Makino et al., “Concentration of live retrovirus with a regenerated cellulose hollow fibre, BMM”, Archives of Virology (1994) 139(1-2):87-96.).

Preferably, the virus is purified using chromatography, such as ion exchange, chromatography. Chromatic purification allows for the production of large volumes of virus containing suspension. The viral product of interest can interact with the chromatic medium by a simple adsorption/desorption mechanism, and large volumes of sample can be processed in a single load. Contaminants which do not have affinity for the adsorbent pass through the column. The virus material can then be eluted in concentrated form.

Preferred anion exchange resins for use in the invention include DEAE, EMD TMAE. Preferred cation exchange resins may comprise a sulfonic acid-modified surface. In one embodiment, the virus is purified using ion exchange chromatography comprising a strong anion exchange resin (e.g. EMD TMAE) for the first step and EMD-SO₃ (cation exchange resin) for the second step. A metal-binding affinity chromatography step can optionally be included for further purification. (See, e.g., WO 97/06243).

A preferred resin for use in the invention is Fractogel™ EMD. This synthetic methacrylate based resin has long, linear polymer chains (so-called “tentacles”) covalently attached. This “tentacle chemistry” allows for a large amount of sterically accessible ligands for the binding of biomolecules without any steric hindrance. This resin also has improved pressure stability.

Column-based liquid affinity chromatography is another preferred purification method for use in the invention. One example of a resin for use in this purification method is Matrex™ Cellufine™ Sulfate (MCS). MCS consists of a rigid spherical (approx. 45-105 μm diameter) cellulose matrix of 3,000 Dalton exclusion limit (its pore structure excludes macromolecules), with a low concentration of sulfate ester functionality on the 6-position of cellulose. As the functional ligand (sulfate ester) is relatively highly dispersed, it presents insufficient cationic charge density to allow for most soluble proteins to adsorb onto the bead surface. Therefore the bulk of the protein found in typical virus pools (cell culture supernatants, e.g. pyrogens and most contaminating proteins, as well as nucleic acids and endotoxins) are washed from the column and a degree of purification of the bound virus is achieved.

The rigid, high-strength beads of MCS tend to resist compression. The pressure/flow characteristics the MCS resin permit high linear flow rates allowing high-speed processing, even in large columns, making it an easily scalable unit operation. In addition a chromatographic purification step with MCS provides increased assurance of safety and product sterility, avoiding excessive product handling and safety concerns. As endotoxins do not bind to it, the MCS purification step allows a rapid and contaminant free depyrogenation. Gentle binding and elution conditions provide high capacity and product yield. The MCS resin therefore represents a simple, rapid, effective, and cost-saving means for concentration, purification and depyrogenation. In addition, MCS resins can be reused repeatedly.

The inactivated virus may be further purified by gradient centrifugation, preferably density gradient centrifugation. For commercial scale operation a continuous flow sucrose gradient centrifugation would be the preferred option. This method is widely used to purify antiviral vaccines and is known to the expert in the field (See J P Gregersen “Herstellung von Virussimpfstoffen aus Zellkulturen” Chapter 4.2 in Pharmazeutische Biotechnology (eds. O. Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.)

The density gradient centrifugation step may be performed using laboratory or commercial scale gradient centrifugation equipment. For example, a swinging bucket rotor, a fixed angle rotor, or a vertical tube rotor, particularly for laboratory scale production of the virus. Preferably, the gradient centrifugation step is performed using a swinging bucket rotor. This type of rotor has a sufficiently long pathlength to provide high quality separations, particularly with multicomponent samples. In addition, swinging bucket rotors have greatly reduced wall effects, and the contents do not reorient during acceleration and deceleration. Because of their longer pathlength, separations take longer compared to fixed angle or vertical tube rotors. The prepared sucrose solutions are controlled via refractometer on their sucrose concentration.

Sucrose gradients for density gradient centrifugation, such as in a swinging bucket centrifuge tubes may be formed prior to centrifugation by the use of a gradient former (continuous/linear). The volume of sample which can be applied to the gradient in a swinging bucket rotor tube is a function of the cross-sectional area of the gradient that is exposed to the sample. If the sample volume is too high, there is not sufficient radial distance in the centrifuge tube for effective separation of components in a multicomponent sample.

An approximate sample volume for swinging bucket rotor SW 28 is 1-5 ml per tube (with a tube diameter of 2.54 cm). The sample is applied to the gradient by pipetting the volume on top of the gradient. The blunt end of the pipette is placed at 45-60° angle to the tube wall, approximately 2-3 mm above the gradient. The sample is injected slowly and allowed to run down the wall of the tube onto the gradient. After centrifugation gradient fractions are recovered by carefully inserting a gauge needle until the bottom of the tube and starting to collect fractions of 2 ml by pumping the liquid from the tube into falcon tubes.

Sucrose density gradients suitable for use with this density gradient centrifugation purification step include 0-60%, 5-60%, 15-60%, 0-50%, 5-50%, 15-50%, 0-40%, 5-40%, and 15-40%. Preferably, the sucrose density gradient is 15-40%, 5-40% or 0-40%.

Alternatively, a discontinuous sucrose density gradient may be used for purification. A discontinuous sucrose density scheme provides for discrete, overlaying layers of differing sucrose concentrations. In one example, a first layer of 50% sucrose is covered by a second layer of 40% sucrose; the second layer is covered by a third layer of 20% sucrose; the third layer is covered by a fourth layer of 10% sucrose; and the fourth layer is covered by the solution containing the virus to be purified.

In one embodiment, inactivated virus is purified by a method comprising a first step of chromatography purification and a second step of gradient centrifugation. Preferably the first step comprises liquid affinity chromatography, such as MCS. Preferably, the second step comprises density gradient centrifugation using a swinging bucket rotor.

Additional purification methods which may be used to purify inactivated SARS virus include the use of a nucleic acid degrading agent, preferably a nucleic acid degrading enzyme, such as a nuclease having DNase and RNase activity, or an endonuclease, such as from Serratia marcescens, commercially available as Benzonase™, membrane adsorbers with anionic functional groups (e.g. Sartobind™) or additional chromatographic steps with anionic functional groups (e.g. DEAE or TMAE). An ultrafiltration/dialfiltration and final sterile filtration step could also be added to the purification method.

Preferably, the purification includes treatment of the SARS viral isolate with one or more nucleic acid degrading enzymes. These enzymes may be used to reduce the level of host cell nucleic acid in the viral purification process. Nucleic acid digesting enzymes for use in cell culture are known in the art and include, for example, Benzonase™.

The treatment of the virus with the nucleic acid degrading enzyme and inactivating agent can be performed by a sequential treatment or in a combined or simultaneous manner. Preferably, the nucleic acid degrading agent is added to the virus preparation prior to the addition of the inactivating agent.

The purified viral preparation of the invention is substantially free of contaminating proteins derived from the cells or cell culture and preferably comprises less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/μg virus antigen, preferably less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/dose. Still more preferably, the purified viral preparation comprises less than about 20 pg, and even more preferably, less than about 10 pg. Methods of measuring host cell nucleic acid levels in a viral sample are known in the art. Standardized methods approved or recommended by regulatory authorities such as the WHO or the FDA are preferred.

The invention includes an inactivated vaccine composition comprising a prophylactically effective amount of SARS viral antigen, preferably spike or an immunogenic fragment thereof. The SARS viral antigen is preferably present in a concentration amount of 0.1 to 50 μg antigen/dose, more preferably 0.3 to 30 μg antigen/dose. Still more preferably, the antigen is about 15 μg/dose.

In one embodiment, a lower concentration of SARS viral antigen is used in inactivated vaccine compositions of the invention. Such lower concentration vaccines may optionally comprise an adjuvant to boost the host immune response to the antigen. In such a “low dose” vaccine, the SARS viral antigen is preferably present in a concentration of less than 15 μg antigen/dose, (i.e., less than 10, 7.5, 5 or 3 μg antigen/dose.

The inactivated vaccine preparations of the invention may further comprise a stabilizer to preserve the integrity of the immunogenic proteins in the inactivated viral preparation. Stabilizers suitable for use in vaccines are known in the art and may include, for example, buffers, sugars, sugar alcohols, and amino acids. Stabilizing buffers are preferably adjusted to a physiological pH range and may include phosphate buffers, Tris buffers, TE (Tris/EDTA), TEN (Tris/NaCl/EDTA) and Earle's salt solution. Stabilizing sugars may include, for example, one or more of saccharose, glucose, fructose, dextranes, dextranesulphate, and trehalose. Stabilizing sugar alcohols may include, for example, Xylite/Xylitole, Mannite/Mannitol, Sorbite/Sorbitol, and Glycerol. Amino acids suitable for use in the invention include, for example, L-glutamine, arginine, cysteine, and lysine. Additional stabilizers which may be used in the invention include Tartaric acid, Pluronic F 68, and Tween 80.

SARS viral isolates which may be used for the inactivated viral preparations of the invention may be obtained and identified by any of the mechanisms described supra. For example, a SARS isolate may be obtained from a clinical sample and plaque purified. Such methods of viral isolation are known in the art.

Further purification procedures can be applied to ensure the seed virus used for preparation of the vaccine does not contain, for example, unwanted adventitious agents. In one embodiment, viral RNA from the viral isolate can be isolated from the virus, purified (and, optionally, the sequence verified through PCR or other means) and then introduced into a suitable cell culture.

As an example of this technique, a clinical viral sample is plaque purified and amplified on vero cells to generate a sufficient amount of the viral sample for analysis. Cellular remnants are then cleared from the supernatant by centrifugation. The virus can then be pelleted by ultracentrifugation and the pellet resuspended in PBS. After further centrifugation purification, the virus containing fraction is treated with a DNase (and optionally also an RNase). Viral RNA is then isolated from this fraction and transfected into a host cell.

Examples 2 and 3 provide an illustration of purification of inactivated whole SARS virus using MCS chromatography resin purification followed by density gradient ultracentrifugation.

Routes and methods of immunization of the vaccines of the invention are discussed in more detail in a section below. Examples 4 and 5 provide illustrations of a mouse immunization scheme with the inactivated SARS virus of the invention.

B. Attenuated SARS Vaccines

The invention includes a composition comprising an attenuated SARS virus. This composition can be used as a prophylactic or therapeutic SARS virus vaccine. Methods of attenuating viruses are known in the art. Such methods include serial passage of the SARS virus in cultured cells (e.g., mammalian cell culture, preferably fetal rhesus kidney cells or VERO cells—see the discussion in Section A above regarding culture of SARS virus), until the SARS virus demonstrates attenuated function. The temperature at which the virus is grown can be any temperature at which with tissue culture passage attenuation occurs. Attenuated function of the SARS virus after one or more passages in cell culture can be measured by one skilled in the art. As used herein, attenuation refers to the decreased virulence of the SARS virus in a human subject. Evidence of attenuated function may be indicated by decreased levels of viral replication or by decreased virulence in an animal model.

Other methods of producing an attenuated SARS virus include passage of the virus in cell culture at sub-optimal or “cold” temperatures and introduction of attenuating mutations into the SARS viral genome by random mutagenesis (e.g., chemical mutagenesis) or site specific directed mutagenesis. Preparation and generation of attenuated RSV vaccines (the methods of which will generally applicable to SARS virus) are disclosed in, for example, EP 0 640 128, U.S. Pat. No. 6,284,254, U.S. Pat. No. 5,922,326, U.S. Pat. No. 5,882,651.

The attenuated derivatives of SARS virus are produced in several ways, such as for example, by introduction of temperature sensitive-mutations either with or without chemical mutagenesis (e.g., 5-fluorouracil), by passage in culture at “cold” temperatures. Such cold adaptation includes passage at temperatures between about 20° C. to about 32° C., and preferably between temperatures of about 22° C. to about 30° C., and most preferably between temperatures of about 24° C. and 28° C. The cold adaptation or attenuation may be performed by passage at increasingly reduced temperatures to introduce additional growth restriction mutations. The number of passages required to obtain safe, immunizing attenuated virus is dependent at least in part on the conditions employed. Periodic testing of the SARS virus culture for virulence and immunizing ability in animals (e.g., mouse, primate) can readily determine the parameters for a particular combination of tissue culture and temperature. The attenuated vaccine will typically be formulated in a dose of from about 10³ to 10⁶ PFU or TCID₅₀, or more for maximal efficacy.

Attenuated virus vaccines for SARS-CoV also are produced by creating virus chimeras comprising sequences derived from at least two different coronaviruses, one of which is a SARS-CoV. For example, a virus chimera is produced that comprises nonstructural protein encoding genes derived from a first coronavirus (e.g., murine, bovine, porcine, canine, feline, avian coronavirus) and one or more structural protein encoding genes (e.g., spike, E, M) from a SARS-CoV. Alternatively, the virus chimera may comprise sequences derived from a human coronavirus that is not a SARS-CoV (e.g., OC43, 229E) together with sequences from a SARS-CoV. Chimeric coronaviruses of the present invention are generated by a variety of methods, including for example allowing for natural RNA recombination in a eukaryotic (e.g., mammalian) cell that contains RNA from each of the parental coronaviruses (e.g., following infection) or by using standard molecular biology techniques known to those of skill in the art to engineer desired virus chimeras (or portions thereof) as cDNA clones, which may then be used to produce infectious virus (see for example, U.S. Pat. No. 6,593,111 B2; Yount et al., 2003, Proc. Natl. Acad. Sci. USA 100(22):12995-13000). An attenuated phenotype of the coronavirus chimeras described herein can be readily measured by one of skill in the art.

Attenuated viruses can be also generated by deleting one or more open reading frames (ORFs) that are not essential for viral replication. Preferably, these deletions occur in the structural region of the genome, such as ORF 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b. See e.g., Haijema B J, Volders H, Rottier P J. J Virol. (2004) 78(8):3863-71; and de Haan, C. A., P. S. Masters, X. Shen, S. Weiss, and P. J. Rottier, “The group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host.” Virology (2002) 296:177-189. Deletion of such regions within a coronavirus such as SARS can be achieved, for example, by reverse genetics or “targeted recombination” (See, e.g., Masters, P. S., “Reverse genetics of the largest RNA viruses”, Adv. Virus Res. (1999) 53:245-264.

Methods of purification of attenuated virus are known in the art and may include one or more of, for instance gradient centrifugation and chromatography. See Gregersen “Herstellung von Virussimpfstoffen aus Zellkulturen” Chapter 4.2 in Pharmazeutische Biotechnology (eds. O. Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.

C. Split SARS Vaccines

The invention includes a composition comprising a split SARS virus formulation and methods for the manufacture thereof. This composition can be used as a prophylactic or therapeutic SARS virus vaccine.

Methods of splitting enveloped viruses are known in the art. Methods of splitting enveloped viruses are disclosed, for example, in WO 02/28422, incorporated herein by reference in its entirety, and specifically including the splitting agents and methods described therein. Methods of splitting influenza viruses are disclosed, for example, in WO 02/067983, WO 02/074336, and WO 01/21151, each of which is incorporated herein by reference in its entirety.

The splitting of the virus is carried out by disrupting or fragmenting whole virus, infectious (wild-type or attenuated) or non-infectious (for example inactivated), with a disrupting concentration of a splitting agent. The disruption results in a full or partial solubilisation of the virus proteins, altering the integrity of the virus.

Preferably, the splitting agent is a non-ionic or an ionic surfactant. Accordingly, the split SARS virus formulations of the invention may also comprise at least one non-ionic surfactant or detergent. Examples of splitting agents useful in the invention include: bile acids and derivatives thereof, non-ionic surfactants, alkylglycosides or alkylthioglycosides and derivatives thereof, acyl sugars, sulphobetaines, betains, polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg, alkylphenoxypolyethoxyethanols, quaternary ammonium compounds, sarcosyl, CTAB (cetyl trimethyl ammonium bromide) or Cetavlon.

Preferably, the ionic surfactant is a cationic detergent. Cationic detergents suitable for use in the invention include detergents comprising a compound of the following formula:

wherein

R₁ , R₂ and R₃ are the same or different and each signifies alkyl or aryl, or

R₁and R₂, together with the nitrogen atom to which these are attached form a 5- or 6-membered heterocyclic ring, and

R₃ signifies alkyl or aryl, or

R₁, R₂ and R₃ together with the nitrogen atom to which these are attached, signify a 5- or 6-membered heterocyclic ring, unsaturated at the nitrogen atom,

R₄ signfies alkyl or aryl, and

X signifies an anion.

Examples of such cationic detergents are cetyltrimethylammonium salts, such as ceytltrimethylammonium bromide (CTAB) and myristyltrimethylammonium salt.

Additional cationic detergents suitable for use in the invention include lipofectine, lipofectamine, and DOT-MA.

Non-ionic surfactants suitable for use in the invention include one or more selected from the group consisting of the octyl- or nonylphenoxy polyoxyethanols (for example the commercially available Triton series), polyoxyethylene sorbitan esters (Tween series) and polyoxyethylene ethers or esters of the general formula: O(CH₂CH₂O)n-A-R

wherein n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or phenyl C₁₋₅₀ alkyl; and combinations of two or more of these.

The invention comprises a method of preparing a split SARS virus comprising contacting the SARS virus with a sufficient amount of splitting agent to disrupt the viral envelope. The loss of integrity after splitting renders the virus non-infectious. Once the disrupted viral envelope proteins are generally no longer associated with whole intact virions, other viral proteins are preferably fully or partially solubilized and are therefore not associated, or only in part associated, with whole intact virions after splitting.

The method of preparing a split SARS virus may further comprise removal of the splitting agents and some or most of the viral lipid material. The process may also include a number of different filtration and/or other separation steps such as ultracentrifugation, ultrafiltration, zonal centrifugation and chromatographic steps in a variety of combinations. The process may also optionally include an inactivation step (as described above) which may be carried out before or after the splitting. The splitting process may be carried out as a batch, continuous, or semi-continuous process.

Split SARS virus vaccines of the invention may include structual proteins, membrane fragments and membrane envelope proteins. Preferably, the split SARS virus preparations of the invention comprise at least half of the viral structural proteins.

One example of a method of preparing a split SARS virus formulation includes the following steps:

(i) propagation of the SARS virus in cell culture, such as MRC-5 cells (ATCC CCL-171), WI-38 cells (ATCC CCL-75), fetal rhesus kidney cells or vero cells (See the discussion in Section A, above, regarding culture of SARS virus);

(ii) harvesting of SARS virus-containing material from the cell culture;

(iii) clarification of the harvested material to remove non-SARS virus material;

(iv) concentration of the harvested SARS virus;

(v) separation of the whole SARS virus from non-virus material;

(vi) splitting of the whole SARS virus using a suitable splitting agent in a density gradient centrifugation step; and

(vii) filtration to remove undesired materials.

The above steps are preferably performed sequentially.

The clarification step is preferably performed by centrifugation at a moderate speed. Alternatively, a filtration step may be used for example with a 0.2 μm membrane.

The concentration step may preferably employ an adsorption method, for instance, using CaHPO₄. Alternatively, filtration may be used, for example ultrafiltration.

A further separation step may also be used in the method of the invention. This further separation step is preferably a zonal centrifugation separation, and may optionally use a sucrose gradient. The sucrose gradient may further comprise a preservative to prevent microbial growth.

The splitting step may also be performed in a sucrose gradient, wherein the sucrose gradient contains the splitting agent.

The method may further comprise a sterile filtration step, optionally at the end of the process. Preferably, there is an inactivation step prior to the final filtration step.

Methods of preparing split SARS virus formulations may further include treatment of the viral formulation with a DNA digesting enzyme. These enzymes may be used to reduce the level of host cell DNA in the viral purification process. DNA digesting enzymes for use in cell culture are known in the art and include, for example, Benzonase®.

Treatment of the SARS virus formulation with a DNA digesting enzyme may occur at any time in the purification and splitting process. Preferably, however, the SARS virus formulation is treated with a DNA digesting enzyme prior to use of a detergent. Still more preferably, the SARS virus formulation is treated with a DNA digesting enzyme, such as Benzonas, prior to treatment with a cationic detergent, such as CTAB.

Methods of purification of split virus are known in the art. See J P Gregersen “Herstellung von Virussimpfstoffen aus Zellkulturen” Chapter 4.2 in Pharmazeutische Biotecnologie (eds. O. Kayser and R H Mueller) Wissenschaftliche Verlagsgesellschaft, Stuttgart, 2000.

The invention includes a split vaccine composition comprising a prophylactically effective amount of SARS viral antigen, preferably spike or an immunogenic fragment thereof. The SARS viral antigen is preferably present in a concentration amount of 0.1 to 50 μg antigen/dose, more preferably 0.3 to 30 μg antigen/dose. Still more preferably, the antigen is about 15 μg/dose.

In one embodiment, a lower concentration of SARS viral antigen is used in split vaccine compositions of the invention. Such lower concentration vaccines may optionally comprise an adjuvant to boost the host immune response to the antigen. In such a “low dose” vaccine, the SARS viral antigen is preferably present in a concentration of less than 15 μg antigen/dose, (i.e., less than 10, 7.5, 5 or 3 μg antigen/dose.

D. Subunit SARS Vaccines

The invention includes a composition comprising an isolated or purified SARS viral antigen or a derivative thereof. The composition may further comprise one or more adjuvants.

SARS viral antigens can be isolated or purified from a SARS virus grown in cell culture. Alternatively, SARS viral antigens can be recombinantly produced by methods known in the art.

The SARS viral antigens used in the invention can be produced in a variety of different expression systems which are known in the art; for example those used with mammalian cells, baculoviruses, bacteria, and yeast. Such expression systems will typically use polynucleotides encoding the viral antigens of the invention. Such sequences can be obtained using standard techniques of molecular biology, including translating the amino acid sequences listed herein. Accordingly, the invention includes polynucleotides encoding for the viral antigens of the invention. In addition, the viral antigens of the invention can be produced (at least in part, preferably in whole) via synthetic chemistry methods.

Insect cell expression systems, such as baculovirus systems, are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insert cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. Similarly, bacterial and mammalian cell expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

A number of appropriate host cells for use with the above systems are also known. For example, mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Mammalian sources of cells include, but are not limited to, human or non-human primate (e.g., MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), fetal rhesus lung cells (ATCC CL-160), human embryonic kidney cells (293 cells, typically transformed by sheared adenovirus type 5 DNA), VERO cells from monkey kidneys), horse, cow (e.g., MDBK cells), sheep, dog (e.g., MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 as described in WO 97/37001), cat, and rodent (e.g., hamster cells such as BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and may be obtained from a wide variety of developmental stages, including for example, adult, neonatal, fetal, and embryo.

Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include, inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Nucleic acid molecules comprising nucleotide sequences of the viral antigens or antibodies of the invention can be stably integrated into a host cell genome or maintained on a stable episomal element in a suitable host cell using various gene delivery techniques well known in the art. See., e.g., U.S. Pat. No. 5,399,346.

Depending on the expression system and host selected, the molecules are produced by growing host cells transformed by an expression vector under conditions whereby the protein is expressed. The expressed protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the product can be purified directly from the media. If it is not secreted, it can be isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

The invention includes a composition comprising an isolated or purified SARS viral antigen or a derivative thereof. The invention also includes a composition comprising at least two isolated or purified SARS viral antigens or derivatives thereof, which have been co-purified or purified separately and then combined. In one embodiment, the SARS viral antigen is a spike (S) protein. In yet another embodiment, the SARS viral antigen is a nucleocapsid (N) protein, a membrane (M) glycoprotein, or an envelope (E) protein. Preferably, the SARS viral antigen is present in the composition in a purity greater than 75% (e.g., 78%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 98%).

The invention includes a vaccine composition comprising a prophylactically effective amount of SARS viral antigen, preferably spike or an immunogenic fragment thereof. The SARS viral antigen is preferably present in a concentration amount of 0.1 to 50 μg antigen/dose, more preferably 0.3 to 30 μg antigen/dose. Still more preferably, the antigen is about 15 μg/dose.

In one embodiment, a lower concentration of SARS viral antigen is used in vaccine compositions of the invention. Such lower concentration vaccines may optionally comprise an adjuvant to boost the host immune response to the antigen. In such a “low dose” vaccine, the SARS viral antigen is preferably present in a concentration of less than 15 μg antigen/dose, (i.e., less than 10, 7.5, 5 or 3 μg antigen/dose.

The following example illustrates a method of preparing a SARS virus spike (S) protein subunit vaccine.

SARS virus S antigen may be isolated and purified from a variety of sources and using a variety of methods, including, but not limited to, S antigen expressed in cultured eukaryotic cells (e.g., mammalian cells, such as VERO, CHO) or bacteria (e.g., E. coli). Expression of may be achieved by a variety of means, such as, for example, from SARS virus infected cell culture or cell culture supernatants, from cultured cells stably transformed with a DNA expression cassette encoding the SARS virus S protein (e.g., RNA polymerase II promoter operably linked to a SARS virus S gene), or from cultured cells infected with a replication-competent or replication-incompetent virus-based expression vector (e.g., adenovirus vector, poxvirus vector, alphavirus vector, retrovirus vector) encoding the SARS virus S protein, as a means to eliminate the need to work with infectious SARS virus.

1. Subunit SARS Vaccines Produced from SARS Virus Cultures

The SARS virus may be grown in cultured mammalian celle, such as VERO cells, then separated from the cultured cells. A SARS viral antigen, such as the S protein, can then be solubilized and separated from the SARS virus, and further isolated and purified.

In one example, the SARS virus may be produced as described in the Inactivated SARS vaccine examples, then the desired SARS antigen, such as spike protein, may be further purified from the end product using techniques known in the art.

In another example, a SARS subunit vaccine may be produced as follows. SARS virus may be produced using a desired mammalian cell line on microcarrier beads in large, controlled fermentors. For example, vaccine quality African Green Monkey kidney cells (VERO cells) at a concentration of 10⁵ cells/mL are added to 60 to 75 L of CMRL 1969 media, pH 7.2, in a 150 L bioreactor containing 360 g of Cytodex-1 microcarrier beads and stirred for 2 hours. Additional CMRL 1969 is added to give a total volume of 150 L. Fetal bovine serum (FBS) is added to a final concentration of 3.5%. Glucose is added to a final concentration of 3.0 g/L and glutamine is added to a final concentration of 0.6 g/L. Dissolved oxygen, pH, agitation and temperature are controlled, and cell growth, glucose, lactate and glutamine levels are monitored. When cells are in logarithmic phases usually on days 3 to 4 reached a density of about 1.0-2.5×10⁶ cells/mL, the culture medium is drained from the fermentor and 120 L of CMRL 1969, pH 7.2 (no FBS) is added and the culture stirred for 10 minutes. The draining and filling of the fermentor is usually repeated once but could be repeated up to three times. After washing the cells, the fermentor is drained and 50 L of CMRL 1969 containing 0.1% (v/v) FBS is added. The SARS virus inoculum is added at a multiplicity of infection (m.o.i.) of 0.001 to 0.01. Trypsin may be added to promote efficient infection. Additional CMRL 1969 with 0.1% FBS is added to give a final volume of 150 L. Incubation is continued at 34 C. One viral harvest is obtained from a single fermentor lot, typically at 2-7 days post-infection. Multiple harvests from a single fermentation may also be obtained.

The isolation and purification of S protein may be effected by a variety of means, as described below. For example, collecting S protein-containing flow-through from ion exchange chromatography of solubilized SARS virus envelope proteins; loading the flow through onto a hydroxyapatite matrix, and selectively eluting the S protein from the hydroxyapatite matrix. The selectively eluted S protein may be further concentrated by tangential flow ultrafiltration.

Alternatively, the isolation and purification may be effected by collecting S protein-containing flow-through from ion exchange chromatography of the solubilized SARS virus envelope proteins; loading the flow through onto a hydroxyapatite matrix and collecting an S protein-containing flow through, selectively removing detergent used in the solubilization step from the hydroxyapatite matrix flow through to provide isolated and purified S protein. The isolated and purified S protein may be subsequently concentrated by tangential flow ultrafiltration

Nucleic acid contaminants may be removed from the isolated and purified S protein by treatment with a nucleic acid degrading agent as described above in the Inactivation section. Preferably, the nucleic acid degrading agent is a nuclease, such as for example, Benzonase.

The isolated and purified S protein may be applied to a gel filtration medium and the S protein subsequently collected therefrom to separate the S protein from contaminants of other molecular weights.

Alternatively, the isolation and purification may be effected by loading S protein on a first ion-exchange medium while permitting contaminants to pass through the medium, eluting the S protein from the first ion-exchange medium, to separate the S protein from contaminants of other molecular weights. The eluted S protein is applied to a second ion-exchange medium while allowing contaminants to pass through the second ion-exchange medium. The S protein is subsequently eluted therefrom, to provide the isolated and purified S protein. The eluted S protein may be concentrated by tangential flow ultrafiltration.

Alternatively, substantially pure SARS virus S protein suitable for use as an immunogen in a subunit vaccine formulation may be prepared from infected cell lysates, such as for example using a non-denaturing detergent buffer containing 1% Triton X-100 and deoxycholate to lyse infected cells. The cell lysates are clarified by centrifugation and S protein is purified from the cell lysates by immunoaffinity purification. A monoclonal antibody against the S protein is generated and coupled to beads and a column is constructed with those beads. SARS-infected cell lysates are applied to the column, and the column is washed with PBS containing 0.1% Triton X-100. Protein bound to the column is eluted with 0.1M glycine, pH 2.5, 0.1% Triton X-100. Elution samples are buffered, such as for example, with Tris, and analyzed for the presence of protein. Fractions containing the protein are pooled and dialyzed against PBS

As discussed above, the present invention includes isolated and purified S protein of SARS virus. In one example, the virus is grown on a vaccine quality cell line, such as VERO cells, and the grown virus is harvested. The virus harvest is filtered and then concentrated typically using tangential flow ultrafiltration using a membrane of desired molecular weight cut-off and diafiltered. The virus harvest concentrate may be centrifuged and the supernatant discarded. The pellet from the centrifugation then is detergent extracted to solubilize the S protein, for example, by resuspending the pellet to the original harvest concentrate volume in an extraction buffer containing a detergent such as a non-ionic detergent including TRITON X-100.

Following centrifugation to remove non-soluble proteins, the S protein extract is purified by chromatographic procedures. The extract may first be applied to an ion exchange chromatography column such as a TMAE-fractogel or S-fractogel column equilibrated to permit the S protein to flow through while impurities are retained on the column.

Next, the flow through may be loaded onto a hydroxyapatite column, equilibrated to permit binding of the S protein to the matrix and to permit contaminants to pass from the column. The bound S protein is then eluted from the column by a suitable elutant. The resulting purified solution of S protein may be further processed to increase its purity. The eluate first may be concentrated by tangential flow ultrafiltration using a membrane of desired molecular weight cut-off. The filtrate may be contacted with a polyethylene glycol of desired molecular weight, for example, about 6000 to 8000, to precipitate the protein. Following centrifugation and discard of the supernatant, the pellet may be resuspended in PBS and dialyzed to remove the polyethylene glycol. Finally, the dialyzed solution of S protein may be sterile filtered. The sterile filtered solution may be adsorbed onto alum. The polyethylene glycol precipitation and resuspension purification step may be effected at an earlier stage of the purification operation, if desired.

Alternatively, SARS virus is recovered following growth and harvesting of the virus, and a concentrate obtained such as, for example using PEG precipitation or tangential flow filtration. The virus is contacted with detergent to solubilize the S proteins. Following centrifugation, the supernatant is recovered to further purification of the S protein and the non-soluble proteins discarded.

The supernatant is applied to an ion exchange chromatography column, such as a TMAE-fractogel or S-fractogel column, suitably equilibrated to permit retention of the S protein on the column. The S protein is eluted from the ion-exchange column under suitable conditions. The eluate then may be passed through a gel filtration column, such as a Sephacryl S-300 column, to separate the S protein from contaminants of other molecular weights. A hydroxyapatite column may be employed in place of the Sephacryl column.

The S protein may be eluted from the column to provide a purified solution of S protein. The eluate may be concentrated by tangential flow ultrafiltration using a membrane of desired molecular weight cut-off. The concentrated S protein solution then may be sterile filtered.

Alternatively, viral harvests may be concentrated by ultrafiltration and the concentrated viral harvests may be subjected to an initial purification step, for example, by gel filtration chromatography, polyethylene glycol precipitation or Cellufine sulfate chromatography. The purified virus may then be detergent extracted to solubilize the S protein. Following solubilization of the S protein, the supernatant may be loaded onto an ion-exchange column such as Cellufine sulfate chromatography column equilibrated to permit the protein to bind to the column while permitting contaminants to flow through. Similarly, a TMAE-fractogel or S-fractogel column may be used in place of the Cellufine sulfate column. The two columns also may be combined in sequential purification steps. The S protein is eluted from the columns to provide a purified solution of the protein. This solution may be concentrated by tangential flow ultrafiltration using a membrane of desired molecular weight cut-off and diafiltered.

Specifically, in one method of S protein purification, the virus harvest concentrate is centrifuged at 28,000×g for 30 minutes at 4 C. The supernatant is discarded and the pellet resuspended in extraction buffer consisting of 10 mM Tris-HCl, pH 7.0, 150 mM NaCl, 2% (w/v) Triton X-100 to the original harvest concentrate volume. Pefabloc is added to a final concentration of 5 mM. The suspension is stirred at room temperature for 30 minutes. The supernatant, containing the soluble S protein, is clarified by centrifugation at 28,000×g for 30 minutes at 4 C. A TMAE--Fractogel column is equilibrated with 10 mM Tris-HCl, pH 7.0, 150 mM NaCl containing 0.02% Triton X-100. The Triton X-100 supernatant, containing the soluble S protein, is loaded directly onto the TRAE-Fractogel column. The total volume added plus 2 bed volumes of 10 mM Tris-HCl, pH 7.0, 150 mM NaCl containing 0.02% Triton X-100 are collected. The TMAE-Fractogel flow-through containing S protein is diluted 3-fold with 10 mM Tris-HCl, pH 7.0, containing 0.02% Triton X-100.

An hydroxyapatite column is equilibrated with 10 mM Tris-HCl, pH 7.0, 50 mM NaCl, 0.02% Triton X-100. After loading the TMAE flow-through, the column is washed with 2 column volumes of 10 mM Tris-HCl, pH 7.0, 50 mM NaCl, 0.02% Triton X-100 followed by 4 column volumes of 5 mM sodium phosphate, pH 7.0, 1M NaCl, 0.02% Triton X-100. The proteins are eluted with 4 column volumes of 20 mM sodium phosphate, pH 7.0, 1M NaCl, 0.02% Triton X-100. Fractions are collected based on A280 and the protein content and antigen concentrations are measured. The purified S protein is ultrafiltered by tangential flow ultrafiltration using a 300 kDa NMWL membrane.

2. Recombinant Production of Subunit SARS Vaccines

As discussed above, SARS virus proteins may be produced by recombinant expression. Host cells suitable for recombinant expression include bacterial, mammalian, insect, yeast, etc. Recombinant expression may be used to produce a full length SARS protein, a fragment thereof, or a fusion therewith.

Fusion peptides may be used to facilitate the expression and purification of the recombinant SARS protein. For example, recombinant production of the SARS polypeptides can be facilitated by the addition a tag protein to the SARS antigen to be expressed as a fusion protein comprising the tag protein and the SARS antigen. Such tag proteins can facilitate purification, detection and stability of the expressed protein. Tag proteins suitable for use in the invention include a polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP-tag,, chitin-binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination anti-terminiantion factor (NusA), E. coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). Preferred tag proteins include His-tag and GST. A full discussion on the use of tag proteins can be found at Terpe et al., “Overview of tag protein fusions: from molecular and biochemical fumdamentals to commercial systems”, Appl Microbiol Biotechnol (2003) 60:523-533.

After purification, the tag proteins may optionally be removed from the expressed fusion protein, i.e., by specifically tailored enzymatic treatments known in the art. Commonly used proteases include enterokinase, tobacco etch virus (TEV), thrombin, and factor X_(a).

Accordingly, the invention further includes a SARS virus subunit vaccine comprising a fusion protein. Preferably, the fusion protein comprises a first amino acid sequence encoded by a SARS virus polynucleotide sequence. SARS virus polynucleotide sequences which may encode said first amino acid sequence include one or more of the SARS virus polynucleotide sequences identified in this application and fragments thereof.

The fusion protein may comprise an amino acid sequence of a SARS virus protein or a fragment thereof. Said SARS virus protein may be selected from one or more of the group consisting of the following SARS virus proteins: P28, P65, Nsp1, Nsp2 (3CL protease), Nsp3, Nsp3, Nsp4, Nsp 5, Nsp6, Nsp 7, Nsp 8, Nsp 9 (RNA polymerase), Nsp 10 (helicase), Nsp 11, Nsp 12, Nsp 13, Spike, Orf 3, Orf 4, Envelope, Matrix, Orf7, Orf8, Orf9, Orf10, Orf11, Nucleocapsid and Orf13.

In one embodiment, the fusion protein comprises a first amino acid sequence comprising a SARS virus antigen or a fragment thereof. Said SARS virus amino acid sequence may comprise one or more of the T-epitope sequences identified above.

Preferably, the fusion protein comprises an amino acid sequence of a SARS virus spike protein, or a fragment thereof. Specific fragments of the spike protein which may be used in the fusion protein include the S1 domain and the S2 domain. Further fragments of the spike protein which may be used in the fusion protein include regions of each of the S1 and S2 domains, including the receptor binding region of the S1 domain, the oligomerization domain regions of the S2 domain, the leucine zipper regions of the S2 domain, the membrane anchor region of the S2 domain, the hydrophobic domain region of the S2 domain, the cystein-rich domain region of the S2 domain, and the cytoplasmic tail region of the S2 domain. (See FIG. 19). Amino acid sequences of the Spike protein corresponding to these regions can be identified by those skilled in the art, including, for example, using the functional predictions set forth earlier in the application (predicted transmembrane helices, predicted N-terminus signaling regions, predicted coiled-coil regions, etc.) as well as by homology comparison to the sequences of other known Coronaviruses (See FIGS. 4F and 5).

The fusion protein may further comprise a second amino acid sequence. Said second amino acid sequence may comprise a polypeptide sequence which facilitates protein expression or purification, preferably one of the tag sequences discussed above. Alternatively, said second amino acid sequence may comprise a second amino acid sequence from a SARS virus. Alternatively, said second amino acid sequence may comprises an amino acid sequence from another virus or bacteria, including one or more of the viruses or bacteria identified in Section I, below.

Said second amino acid sequence may comprise an amino acid sequence from another respiratory virus. Said second amino acid sequence may comprise an amino acid sequence from a virus selected from the group consisting of coronavirus, influenza virus, rhinovirus, parainfluenza virus (PIV), respiratory syncytial virus (RSV), adenovirus, and metapneumovirus.

In one embodiment, said second amino acid sequence may comprise an amino acid sequence from an adjuvant, including one or more of the adjuvants identified in section I, below.

In one embodiment, the invention includes a fusion protein comprising an amino acid sequence of a SARS virus spike protein or a fragment thereof. The fusion protein may further comprise a second amino acid sequence comprising an amino acid sequence selected from the group consisting of a second SARS virus protein, a non-SARS virus protein, a bacterial protein, and an adjuvant.

(a) Bacterial Expression of Subunit SARS Vaccines

In one embodiment, bacterial host cells are used for recombinant expression of SARS virus proteins. Bacterial host cells suitable for use in the invention include, for example, E. coli, Bacillus subtilis, and Streptococcus spp.

The SARS viral protein may be modified to facilitate bacterial recombinant expression. In particular, the SARS spike protein may be modified to facilitate transport of the spike protein to the surface of the bacterial host cell.

Applicants have discovered that there is strong structural homology between the SARS virus spike protein and the NadA protein of Neisseria meningitidis. Both proteins have an N-terminal globular “head” domain (amino acids 24-87), an intermediate alpha-helix region with high propensity to form coiled-coil structures (amino acids 88-350), and a C-terminal membrane anchor domain formed by four amphipatix transmembrane beta strands (amino acids 351-405 of NadA). In addition, a leucine zipper motive is present within the coiled-coil segment. See, FIG. 19 depicting the SARS spike protein structure Comanducci et al., “NadA, a Novel Vaccine Candidate of Neisseria meningitidis”, J. Exp. Med. 195 (11): 1445-1454 (2002). In addition, a leucine zipper motif of NadA is present within the coiled-coil segment. The NadA protein also forms high molecular weight surface-exposed oligomers (corresponding to three or four monomers) anchored to meningococcal outer membrane.

When the NadA protein is expressed in E. coli, the full-length protein is assembled in oligomers anchored to the outer membrane of E. coli, similar to the way the protein is presented in meningococcus. The NadA protein devoid of the predicted membrane anchor domain is then secreted into the culture supernatant. This secreted protein is soluble and still organized in trimers.

The invention therefore includes a fusion protein comprising an amino acid sequence of a SARS virus spike protein or a fragment thereof and a second amino acid sequence of a bacterial adhesion protein or a fragment thereof. Preferably, said adhesion protein is selected from the group consisting of NadA, YadA (of enteropathogenic Yersinia), and UspA2 (of Moraxella catarrhalis). Additional NadA-like proteins include serum resistance protein DsrA of Haemophilus ducreyi, the immunoglobulin binding proteins EibA, C, D, and F of E. coli, outer membrane protein 100 of Actinobacillus actinomycetemcomitans, the saa gene carried on the large virulence plasmid present in shiga toxigenic strains of E. coli (STEC), and each of the bacterial adhesion proteins described in U.K. Patent Application No. 0315022.4, filed on Jun. 26, 2003, each of which are specifically incorporated herein by reference.

Preferably, said adhesion protein comprises NadA or a fragment thereof.

Such fusion proteins may be used to facilitate recombinant expression of immunogenic portions of SARS surface antigens, such as spike. These fusion constructs may also allow the SARS S1 and/or S2 domains to adapt to a native confirmation. These fusion proteins are also able to oligomerize and form dimers or trimers, allowing the S1 and/or S2 domains to associate and adapt conformations as in the native SARS spike protein. Further, these expression constructs facilitate surface exposure of the SARS spike protein.

The fusion proteins of the invention preferably comprise a leader peptide from a NadA like protein, preferably NadA, a polypeptide from the immunogenic “head” region of the spike protein, and a stalk region from either the NadA like protein or the Spike protein. During expression and processing of the fusion protein, one or more amino acids may be cleaved off or removed, such as, i.e., the leader peptide or a membrane anchor domain.

The stalk regions facilitate oligomerization of the expression protein. Optionally, the fusion proteins of the invention further include an anchor region of a NadA like protein. This anchor region allows the expression fusion protein to anchor and assemble on the bacterial cell surface.

The fusion proteins of the invention include the following constructs:

(i) the NadA leader peptide (optionally also including the first 6 amino acids of the mature NadA protein to facilitate processing of the leader peptide and appropriate maturation of the protein) followed by the Spike S1 domain. Preferably, this construct comprises amino acids 1-29 of NadA (corresponding to the NadA leader peptide and the first 6 amino acids of the mature NadA protein, as shown in FIG. 22 and as set forth below) followed by amino acids 14-662 of a SARS virus Spike protein (corresponding to the S1 domain, see FIG. 19 and SEQ ID NO: 6042 and as set forth below). Specifically, construct (i) comprises SEQ ID NO: 7302.

(ii) the NadA leader peptide (optionally also including the first 6 amino acids of the mature NadA protein to facilitate processing of the leader peptide and appropriate maturation of the protein) followed by the Spike S1 domain, followed by the stalk and anchor membrane domains of NadA. Preferably, this construct comprises amino acids 1-29 of NadA (corresponding to the NadA leader peptide and the first 6 amino acids of the mature NadA protein, as shown in FIG. 22 and as set forth below) followed by amino acids 14-662 of a SARS virus Spike protein (corresponding to the S1 domain, see FIG. 19 and SEQ ID NO: 6042 and as set forth below) followed by amino acids 88-405 of NadA (corresponding to the stalk and the anchor membrane domains). Specifically, construct (ii) comprises SEQ ID NO: 7303.

(iii) the NadA leader peptide (optionally also including the first 6 amino acids of the mature NadA protein) followed by a SARS virus Spike S1 domain, followed by the NadA stalk domain. Preferably, this construct-comprises amino acids 1-29 of NadA followed by amino acids 14-662 of a SARS virus Spike protein (corresponding to the S1 domain), followed by amino acids 88-350 of NadA (corresponding to the stalk domain). Specifically, construct (iii) comprises SEQ ID NO: 7304.

(iv) the NadA leader peptide (optionally also including the first 6 amino acids of the mature NadA protein), followed by a SARS virus Spike S1 and S2 domain (excluding the putative transmembrane region), followed by the anchor domain of NadA. Preferably, this construct comprises amino acids 1-29 of NadA, followed by amino acids 14-1195 of a SARS virus Spike protein (corresponding to S1 and S2, excluding the putative transmembrane region), followed by amino acids 351-405 of NadA (corresponding to the NadA anchor domain). Specifically, construct (iv) comprises SEQ ID NO: 7305. Alternatively, the NadA anchor domain may comprise amino acids 332-405 of NadA.

(v) the NadA leader peptide (optionally also including the first 6 amino acids of the mature NadA protein), followed by a SARS virus Spike S1 and S2 domain (exclusing the putative transmembrane region). Preferably, this construct comprises amino acids 1-29 of NadA, followed by amino acids 14-1195 of a SARS virus Spike protein. Specifically, construct (v) comprises SEQ ID NO: 7306.

In each of constructs (i) to (v), the first 23 amino acids are the NadA leader peptide, and the GS dipeptide at residues 679-680 arises from the insertion of a restriction enzyme site.

In constructs (i), (ii) and (iii), the NadA “head” is replaced by the Spike S1 domain, and the fusion proteins are anchored to the outer membrane of E. coli or secreted in the culture supernatant, respectively. In constructs (iv) and (v), the “head” and “stalk” domains of NadA are replaced by S1 and S2 Spike domains; also in this case, the two fusion proteins are anchored to the outer membrane of E. Coli or secreted in the culture supernatant, respectively.

Accordingly, the invention further includes a fusion protein comprising an amino acid sequence of a SARS virus spike protein or a fragment thereof and a second amino acid sequence of a bacterial adhesion protein or a fragment thereof. Preferably, amino acids corresponding to the “head” of the adhesion protein are replaced by amino acids corresponding to a SARS virus Spike S1 domain. Alternatively, the amino acids corresponding to the “head” and “stalk” domains of the bacterial adhesion protein are replaced by amino acids corresponding to the SARS virus spike protein S1 and S2 domains.

As discussed above and shown in FIG. 19, the S1 domain of the Spike protein is identified as the globular receptor binding “head” region. The S1 domain of the Spike protein preferably comprises about amino acids 14-662 of SEQ ID NO: 6042. The S1 domain may comprise a shorter amino acid sequence, wherein amino acids are removed from either the N-terminal or C-terminal regions. Preferably, 3, 5, 7, 9, 13, 15, 20 or 25 amino acids are removed from either the N-terminal or C-terminal regions. The S1 domain further includes amino acid sequences having sequence identity to the S1 region of SEQ ID NO: 6042. An example of the S1 domain is SEQ ID NO: 7307:

As discussed above and shown in FIG. 19, the S2 domain of the Spike protein is identified as the “stalk” region. The “stalk” region comprises oligomerization domain regions, a leucine zipper domain regions, membrane anchor regions, hydrophobic domain regions, cystein-rich domain region and a cytoplasmic tail region. The S2 domain of the Spike protein preferably excludes the transmembrane region and comprises about amino acids 663-1195 of SEQ ID NO: 6042. The S2 domain may comprise a shorter amino acid sequence, wherein amino acids are removed from either the N-terminal or C-terminal regions. Preferably, 3, 5, 7, 9, 13, 15, 20 or 25 amino acids are removed from either the N-terminal or C-terminal regions. The S2 domain further includes amino acid sequences having sequence identity to the S2 region of SEQ ID NO: 6042. An example of the S1 domain (with the transmembrane region excluded) is SEQ ID NO: 7308.

An example of the NadA protein described above is SEQ ID NO: 7309. As discussed above, the leader sequence of NadA used in the fusion protein preferably comprises about the first 29 amino acids of NadA (including a leader sequence with about 6 amino acids of the NadA head protein). Examples of such a leader sequences are set forth as SEQ ID NOS: 7310 and 7311 below. The fusion protein may use a leader sequence comprising a shorter amino acid sequence, wherein amino acids are removed from either the N-terminal or C-terminal regions. Preferably, 1, 2, 3, 4, or 5 amino acids are removed from either the N-terminal or C-terminal end of the sequence. The leader sequence used in the fusion protein may also include an amino acid sequences having sequence identity to SEQ ID NO: 7310 or SEQ ID NO: 7311. Preferably, the leader sequence comprises SEQ ID NO: 7311.

Optionally, the fusion peptide comprises about the first 6 amino acids of the mature NadA protein to facilitate processing of the leader peptide and appropriate maturation of the protein. An examples of the first 6 amino acids of a mature NadA proteins is SEQ ID NO: 7312.

As discussed above, the stalk and anchor sequences of NadA used in the fusion protein preferably comprise about amino acids 88-405 of NadA. An example of an amino acid sequence comprising NadA stalk and anchor regions is set forth below as SEQ ID NO: 7313 below. An example of an amino acid sequence comprising a NadA stalk region (without the anchor region) is set forth as SEQ ID NO: 7314 below. An example of an amino acid sequence comprising a NadA anchor region is set forth as SEQ ID NO: 7315 below. The fusion protein may use a stalk (and/or anchor) sequence comprising a shorter amino acid sequence, wherein amino acids are removed from either the N-terminal or C-terminal regions. Preferably, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids are removed from either the N-terminal or C-terminal end of the sequence. The leader sequence used in the fusion protein may also include an amino acid sequences having sequence identity to the SEQ ID NO: 7313.

The fusion proteins of the invention, including those described above, may be prepared, for example, as follows. Single fragments (such as the regions described above) may be amplified by PCR using the oligonucleotide primers set forth in the Table below. (S1_(L) refers to the Spike protein fused to the leader peptide of NadA; S2 refers to the stalk region of the Spike protein, with and without the stop codon). The oligonucleotides were designed on the basis of the DNA sequence of NadA from N. meningitidis B 2996 strain and of Spike from SARS virus isolate FRA1. Each oligonucleotide includes a restriction site as a tail in order to direct the cloning into the expression vector pET21b. SEQ ID NO: Restriction site S1_(L) For 7316 NdeI S1_(L) Rev 7317 BamHI S2 For 7318 BamHI S2 Rev 7319 HindIII S2-stop Rev 7320 XhoI NadA₈₈ For 7321 BamHI NadA₃₅₀ Rev 7322 XhoI NadA₃₃₂ For 7323 HindIII NadA₄₀₅ Rev 7324 XhoI

The single fragments are sequentially cloned into pET21b vector, in order to express the proteins under the control of inducible T7 promoter. The S1 domain of the Spike protein fused to the leader peptide of NadA (S1_(L)) was obtained by PCR using the primers S1_(L)-For and S1_(L)-Rev. The forward oligonucleotide primer contains the NdeI restriction sequence and the sequence coding for the leader peptide of NadA plus the first 6 aminoacids of the mature protein. The PCR fragment was cloned as a NdeI/BamHI fragment in the pET21b vector opened with the same restriction enzymes. This clone (PET-S1_(L)) was then used to sequentially clone the other different domains, as BamHI/XhoI, BamHI/HindIII or HindIII/XhoI fragments. BamHI and HindIII restriction sites introduce the aminoacids GS and KL, respectively.

The PCR amplification protocol was as follows: 200 ng of genomic DNA from Neisseria meningitidis 2996 or 10 ng of plasmid DNA preparation (plasmid pCMVnew, containing the entire gene coding of the Spike protein), were used as template in the presence of 40 μM of each oligonucletide primer, 400-800 μM dNTPs solution, 1× PCR buffer (including 1.5 mM MgCl₂), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ or Invitrogen Platinum Pfx DNA polymerase).

After a preliminary 3 minute incubation of the whole mix at 95° C., each sample underwent a two-step amplification: the first 5 cycles were performed using the hybridisation temperature that excluded the restriction enzyme tail of the primer (Tm1). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (Tm2). Elongation times, performed at 68° C. or 72° C., varied according to the length of the fragment to be amplified. The cycles were completed with a 10 minute extension step at 68° C. or 72° C.

The amplified DNA was either loaded directly on agarose gel and the DNA fragment corresponding to the band of correct size was purified from the gel using the Qiagen™ Gel Extraction Kit, following the manufacturer's protocol.

The purified DNA corresponding to the amplified fragment and the plasmid vectors were digested with the appropriate restriction enzymes, purified using the QIAquick™ PCR purification kit (following the manufacturer's instructions) and ligation reactions were performed.

The ligation products were transformed into competent E. coli DH5α and screening for recombinant clones was performed by growing randomly-selected colonies and extracting the plasmid DNA using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions.

Recombinant plasmids were introduced into E. coli BL21(DE3) used as expression host. Single recombinant colonies were inoculated into LB+ ampicillin and incubated at 37° C. for 14-16 h. Bacteria were directly recovered by centrifugation (uninduced conditions) or diluted in fresh medium and grown at 37° C. until OD₆₀₀ between 0.4-0.8. Protein expression was induced by addition of 1 mM Isopropyl-1-thio-β-D-galactopyranoside (IPTG) for three hours (induced conditions).

Whole cell lysates were obtained resuspending bacteria in SDS-sample buffer 1× and boiling for 5-10 min. Equal amounts of proteins were separated using NuPAGE (Invitrogen) or BIORAD Gel System, according to the manufacturer's instructions. Proteins were revealed by Coomassie-blue staining or transferred onto nitrocellulose membranes for western blot analysis. Western blot was performed using a rabbit polyclonal anti-serum against purified NadA_(Δ351-405) (diluted 1:3000) and a secondary peroxidase-conjugate antibody (DAKO).

Results of the expression in E. coli of S1_(L), S1_(L)-NadA and S1_(L)-Nad_(Δanchor) are shown in FIGS. 38 and 39. Schematics of the fusion constructs are shown in FIG. 37.

Bacterial expression of the SARS viral antigens may also be used to prepare compositions comprising outer membrane vesicles wherein said outer membrane vesicles comprise one or more SARS viral antigens.

Outer Membrane Vesicles (“OMV”), also referred to as blebs, refer to vesicles formed or derived from fragments of the outer membrane of a Gram negative bacterium. OMVs typically comprise outer membrane proteins (OMPs), lipids, phospholipids, periplasmic material and lipopolysaccharide (LPS). Gram negative bacteria often shed OMVs during virulent infections in a process known as blebbing. OMVs can also be obtained from Gram negative bacteria via a number of chemical denaturation processes, such as detergent extraction. Synthetic OMVs or liposomes, comprising a lipid bilayer and typically enclosing an aqueous core, can also be prepared with the SARS viral antigens of the invention.

The OMVs of the invention are preferably lipid vesicles comprising a lipid bilayer surrounding an aquous core. Typically the lipid vesicles are of unilamellar structure (i.e., a single lipid bilayer surrounds the aquous core), although multilammelar lipid vesicles may also be used in the compositions of the invention. OMVs typically have sizes in the nanomolar to micromolar range, e.g., from 1 nM to 100 μM, more typically from 10 nM to 10 μM and preferably from 30 nM to 1 μM.

The OMVs of the invention are preferably prepared from gram negative bacteria. Gram negative bacteria are those bacteria that fail to resist decolorization in the commonly known Gram staining method. Gram negative bacteria are characterized by a complex multilater cell wall and often possess an outer layer polysaccharide capsule. Gram negative bacteria suitable for producing OMVs include, for example, species from Neisseria, Moraxella, Kingella, Acinetobacter, Brucella, Bordetella, Chlamydia, Porphyromonas, Actinobacillus, Borelia, Serratia, Campylobacter, Helicobacter, Haemophilus, Escherichia, Legionella, Salmonella, Pseudomonas and Yersinia.

The OMVs of the invention preferably comprise one or more SARS viral antigens or a fragment thereof. The SARS viral antigens may be recombinantly expressed in a Gram negative bacterial host cell and then harvested with the OMV.

Antigenic components, such as recombinantly expressed SARS viral antigens, may be located in any or all of the three main compartments of the lipid vesicles, including attached to either the interior or exterior surface of the lipid vesicle, for example via a membrane anchor domain, or attachment to a lipid moiety; inserted into the lipid bilayer, for example where the antigenic component is itself a hydrophobic or lipid based entity; or located within the aqueous center or core of the lipid vesicle.

Synthetically prepared OMVs, or liposomes, may be used in the invention. Such liposomes may comprise a number of different lipids and fatty acids. Suitable lipids for inclusion in liposomes of the invention include but are not limited to phophatidylinositol-(4,5)-diphosphate, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidyglycerol, cholesterol, beta-oleolyl-gamma-palmitoyl, lipopolysaccharides and galactocerbrosides.

Suitable means for extraction of OMVs from bacterial sources include deoxycholate extraction, Tris/HCl/EDTA extraction, and lithium acetate extraction. Preferably, the extraction process comprises a physical and/or chemical means to disrupt the bacterial cell outer membrane in order to release sufficient OMVs for purification and isolation. See, e.g., WO 03/051379.

The OMVs of the invention may be enriched and/or supplemented with antigenic components, such as SARS viral antigens, by methods known in the art, including, for example, direct combination in vitro where an energetic combination step can optionally be applied to facilitate integration of the antigenic component into a compartment of the liposome. Methods of energetic combination suitable for use in the invention include homogenization, ultrasonication, extrusion, and combinations thereof.

Preferably, the antigenic component, such as the SARS viral antigen, is recombinantly produced by the host cell from which the OMV is derived. In one embodiment, such OMVs are prepared by introducing nucleic acid sequence encoding for the SARS viral antigen into the recombinant host cell. Preferably the nucleic acid sequence encoding for the SARS viral antigen is controlled by a strong promoter sequence. Preferably, the nucleic acid sequence encoding the SARS viral antigen further comprises an outer-membrane targeting signal. For example, the nucleic acid sequence encoding the SARS viral antigen may be fused to a sequence encoding for a naturally occurring outer membrane protein of the bacterial host. Preferably, the nucleic acid sequence encoding the SARS viral antigen is fused to the signal peptide sequence of the naturally occurring outer membrane protein of the bacterial host.

Methods of preparing an optimizing OMVs for use in vaccines are disclosed in, for example Filip et al., J. Bact. (1973) 115: 717-722; Davies et al., J. Immunol. Method (1990) 143:215-225; and WO 01/09350.

In one embodiment, a bacterial host cell, such as E. coli, are transformed to express the SARS spike protein. As discussed above, the spike protein may be modified to facilitate bacterial expression and transport of the spike protein to the surface of the host cell. Each of the Spike/NadA fusion constructs discussed above may be used in the OMV preparations of the invention. Preferably, constructs comprising the spike S1 globular head domain fused to the stalk region of NadA are used to generate OMVs. The construct may optionally include the NadA leader peptide as well as the NadA anchor peptide. Schematic diagrams of these preferred OMV constructs are depicted in FIG. 49.

Example 6 describes one method of preparing the OMVs of the invention.

(b) Mammalian Expression of Subunit SARS Vaccine

As discussed above, mammalian host cells may be used for recombinant expression of SARS virus proteins. Mammalian host cells suitable for use in the invention include, for example, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Mammalian sources of cells include, but are not limited to, human or non-human primate (e.g., MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), human embryonic kidney cells (293 cells, typically transformed by sheared adenovirus type 5 DNA), VERO cells from monkey kidneys (including, for example COS7 cells), horse, cow (e.g., MDBK cells), sheep, dog (e.g., MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 as described in WO 97/37001), cat, and rodent (e.g., hamster cells such as BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and may be obtained from a wide variety of developmental stages, including for example, adult, neonatal, fetal, and embryo.

The polynucleotides encoding the SARS viral proteins may be modified to facilitate or enhance expression. For example, commercial leader sequences known in the art, such as tPA or IgK or interleukin-2, may be used in the recombinant constructs. Preferably, however, the natural SARS leader sequence is used. Use of the natural leader sequence can be used to ensure that the protein will be trafficked in human cells in the same way as during a normal viral infection, which may be advantageous e.g. for DNA vaccines, where antigen is expressed in situ.

As discussed above, tag sequences can be used in the expression constructs to facilitate purification, detection and stability of the expressed protein. Tag proteins suitable for use in the invention include a polyarginine tag (Arg-tag), polyhistidine tag (His-tag), FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding peptide, cellulose-binding domain, SBP-tag, chitin-binding domain, glutathione S-transferase-tag (GST), maltose-binding protein, transcription termination anti-terminiantion factor (NusA), E. coli thioredoxin (TrxA) and protein disulfide isomerase I (DsbA). Preferred tag proteins include His-tag and GST. A full discussion on the use of tag proteins can be found at Terpe et al., “Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems”, Appl Microbiol Biotechnol (2003) 60:523-533.

After purification, the tag proteins may optionally be removed from the expressed fusion protein, i.e., by specifically tailored enzymatic treatments known in the art. Commonly used proteases include enterokinase, tobacco etch virus (TEV), thrombin, and factor X_(a).

One or more amino acid sequences or amino acid domains of the spike protein may be removed to facilitate mammalian recombinant expression. For instance, the entire S2 domain or the spike transmembrane region may be removed. Representative examples of some expression constructs of both full length and truncated spike glycoprotein suitable for mammalian expression are shown in FIG. 40. Polynucleotide sequences representing each construct are shown in SEQ ID NOS 6578-6583. A description of each annotation is shown below: Clone Expression Name Description Construct nSh natural leader sequence SEQ ID NO: 6578 full length Spike histidine tag nS natural leader sequence SEQ ID NO: 6579 full length Spike nShΔTC natural leader sequence SEQ ID NO: 6580 Spike without transmembrane sequence histidine tag nSΔTC natural leader sequence SEQ ID NO: 6581 Spike without transmembrane sequence nS1h natural leader sequence SEQ ID NO: 6582 S1 domain histidine tag nS1 natural leader sequence SEQ ID NO: 6583 S1 domain

Cloned cDNA fragments that encompass full-length Spike coding sequences, as well as a Spike construct deleted of the transmembrane and cytoplasmic domains (TM-Cy-deleted Spike) for secretion were inserted into an expression vector pCMVIII to create nSh and nShΔTC, respectively. Both spike proteins were tagged with six histidine residues at the end of C-terminus to aid initial characterization of the expressed spike proteins. Similar sequences encoding full-length Spike or transmembrane and cytoplasmic domain deleted Spike, but without the histidine “tag” are readily substituted by one of skill in the art.

The likely locations of the expressed spike constructs was assessed by separating expressed proteins into an aqueous fraction (AF) and a detergent fraction (DF) using the procedure shown in FIG. 48, with results of western blot analysis shown in FIG. 43. The above described vector constructs were evaluated for expression after transfection into COS7 cells. The construct expressing the full length spike protein remained in the cell membrane while the construct expressing the truncated spike protein was located either in the cytosol (FIG. 43) or secreted into the cell medium (FIG. 44). As shown in FIG. 43, full-length spike protein is found in DF (membrane) in an aggregated form, while the truncated protein is found in AF (cytosol) as a monomer. As shown in FIG. 44, deleted proteins (ShΔTC) are secreted, and a small fraction of full-length spike protein is detected in the medium by rabbit serum.

Recombinantly expressed spike proteins may be oligomerized. When the spike proteins are to be used in a vaccine or to generate antibodies specific to the spike protein, they are preferably oligomerized. In order to obtain oligomerized spike protein, it is preferred to maintain the transmembrane domain in the recombinant expression construct. For example, FIG. 41 illustrates a western blot of COS7 cell lysates comparing expressed nSh and nShΔTC using both anti-his tag and rabbit anti-SARS antibodies. As shown full-length (nSh) aggregates, but the truncated (nShΔTC) spike protein does not. Antibody raised against the His-tagged protein recognizes full-length and truncated spike proteins in native and reduced forms. Rabbit antiserum recognizes spike protein only in non-reducing conditions. Spike aggregates or oligomers were present in larger amounts in the cell lysates from the expressed nSh constructs. Preferably, the oligomerized spike proteins form a homotrimer, as indicated in FIG. 47

A further experiment, illustrated in FIG. 42, demonstrates that the oligomerization of the expressed nSh constructs is likely due to a non-covalent linkage (and is likely not due to, for example, a disulfide bond). The oligomer dissociates into monomers at elevated temperature (80-100° C.), but is stable in reducing conditions if not heated.

It is further preferred that recombinantly expressed spike proteins are glycoslyated. Tunicamycin and glycosidases were used to assess glycosylation. FIG. 45 illustrates that glycoslation of expressed spike proteins is not affected by removal of the transmembrane domain region. Both full-length (Sh) and truncated (ShΔTC) SARS spike proteins are glycosylated.

Preferably, expression of the constructs of the invention is not toxic to the mammalian host cell. FIG. 46 demonstrates that expression of the illustrated spike constructs is not toxic to the COS7 host cell.

Methods for transfecting, expressing, culturing, isolating and purifying recombinant proteins from mammalian cell cultures are known in the art. For example, the SARS spike constructs of the invention may be expressed in 293 cells. These cells may be cultured and transfected in static or monolayer cultures. For rapid large-scale production of SARS protein antigens in sufficient quantities for in vitro and in vivo evaluation, including immunogenicity studies, large-scale transient transfection of 293 (human embryonic kidney) cells may be used to obtain milligram quantities of the recombinant antigen(s). Alternatively, larger scale transfection of these cells may be performed with 293 cells in suspension culture. Preferably, the expressed SARS proteins are harvested from the transfected cells between 48 and 72 hours after transfection or even from 72 to 96 or more hours after transfection.

Where the host cells are transfected with truncated spike expression constructs, the expressed spike protein is secreted from the host cells and collected from the cell media. After concentration, the spike protein may be purified from the media using, for example, GNA lectin followed by DEAE and ceramic hydroxyapatite column chromatography.

Where the host cells are transfected with full length spike expression constructs, but rather is retained within the cells, and may be purified from triton X-100 detergent extracted cells. The full-length Spike protein can then be captured on GNA lectin, followed by hydroxyapatite and SP chromatography.

Chinese Hamster Ovary (CHO) or other eukaryotic (e.g., mammalian) cells that stably express the SARS viral antigens of the invention may also be derived (e.g. FIG. 73). Preferably, the cells are CHO cells, and these constructs will comprise one or more marker or selection genes in order to select for the desired CHO cells. In one embodiment, the constructs comprise a CMV enhancer/promoter, ampicillin resistance gene, and a fused DHFR and attenuated neomycin gene for selection purposes. Stable cell lines can then be produced using the neomycin selection system in CHOK-1 cells. Selected clones can then be sequenced to verify the integrity of the insert, and transient transfections can then be performed using Trans-LT1 polyamine transfection reagent (PanVera Corp., Madison, Wis.) to assess the expression level and also the integrity of the expressed protein by ELISA and western blot analysis.

Methods for derivation of CHO cells stably expressing the SARS viral antigens of the invention comprise the steps of transfection and primary screening with selective medium. Optionally, these steps are followed by subcloning to assure purity of cell lines. Cell culture supernatants can be assayed using an antigen capture ELISA to quantify expression levels at all stages of selection and amplification.

For full-length Spike expression constructs, methanol fixed cells can be screened for internal expression by immunofluorescent staining using a rabbit anti-SARS antibody. Successive measurements at the T75-flask stage of expansion can be employed to assure stability of expression levels. The molecular mass and integrity of the expressed proteins can be checked by PAGE both under native and reducing and denaturing conditions, followed by immunoprobing.

In one embodiment, the pCMV3 vectors expressing SARS-CoV Spike proteins in either full-length or truncated forms is introduced into CHOK-1 cells using the Trans-LT-1 reagent. On day one, 1×10⁶ cells are plated on 100 mm dishes in non-selective F12 media+10% Fetal Bovine Serum+4 mM Glutamine. On day two, the cells are transfected with a DNA:LT-1 mixture and the media then replaced with complete F12 media. Twenty-four to forty-eight hours later depending on the cell density, each 100 mm dish is split to 4-6 100 mm dishes. The medium is changed to complete selective media containing Geneticin (neomycin) at 500 μg/ml. All bovine serum used in these procedures is from TSE-free sources that meet current FDA standards. Twenty-four hours later the medium is changed to complete selective medium plus 500 ug/ml neomycin. Ten to fourteen days later, individual colonies are picked and transferred to 96 well plates and cultured in complete selective medium but without G418. When approximately 80% of the wells are confluent, twenty-four hour supernatants are screened by spike capture ELISA positive clones are transferred to twenty-four well plates. For the initial expression of full length Spike protein, methanol fixed cells will be screened by immunoflourescent staining using a rabbit anti-SARS antibody. After the low expressing cell lines have been eliminated and there are less than 20-30 cell lines, capture ELISA and westerns will be used to determine the expression level after cell lysis. A portion of each cell line will be pelleted, weighed and lysed in 1% triton lysis buffer containing MOPS, NaCl and MgCl₂ at the same ratio of cell weight to lysis buffer. After lysis the supernatant is collected and expression level is determined. Three to four clones producing the highest levels of spike protein in correct structure and conformation will be grown in three-liter bioreactors for expansion and adaptation to low serum suspension culture conditions for scale-up.

The antigen capture ELISA assay for the SARS spike protein can be performed as described in the art. A brief description of this assay follows. 96 well flat-bottom plates (Corning, Corning, N.Y.) are coated with 250 ng per well of purified immunoglobulin obtained from rabbit sera that were immunized with inactivated SARS virus. Between steps, the plates are washed in a buffer containing 16% NaCl and 1% Triton X100. 100 μL of supernatant or lysate samples (diluted in a buffer containing 100 mM NaPO₄, 0.1% Casein, 1 mM EDTA, 1% Triton X100, 0.5M NaCl and 0.01% Thiomersal, pH 7.5) are added and incubated for 2 hours at 37° C. Bound antigen is reacted against pooled SARS+ve serum or high affinity monoclonal antibody either human or mouse against SARS spike protein (1 hour incubation, 37° C.) and detected using appropriate species-specific peroxidase conjugated second antibody (30 minute incubation at 37° C.; TAGO, Burlingame, Calif.). The plates are developed for 15 minutes at room temperature using TMB substrate (Pierce, Rockford, Ill.) and the reaction stopped using 4N phosphoric acid. The plates are read at a wavelength of 450 nm and the concentration of protein per ml sample is derived from a standard curve (OD vs. protein concentration) based on serial dilutions of a known concentration of recombinant spike protein.

The immunoprobing analysis can also be performed following the standard methods described elsewhere in the art. A brief description follows. 10-20 μl of the sample is analyzed on 4-20% SDS PAGE under non-reducing/denaturing conditions with mild heating. The gels are run for 1.5-2.0 hours at 100V constant voltage. The proteins are then transferred onto nitrocellulose membranes (Millipore, Bedford, Mass.) for 45 min using the semidry western transfer system (BioRad, Hercules, Calif.) following the manufacturer's instructions. The membrane is then reacted against polyclonal anti-spike rabbit serum, followed by anti-rabbit Ig conjugated to Alexa 688 (Molecular Probes, Oregon). The blots are scanned using an infrared imaging system (LI-Cor, Inc., Lincoln, Nebr.).

The highest expressing candidate cell lines can be screened for spike protein expression and stability in small-scale (3 liter) suspension cultures. The candidate clone can be further evaluated for level of expression as well as integrity of expressed protein after amplification, and subsequently tested for expression stability in the absence of selection. The selected clones can also be tested for maintenance of the DNA sequence integrity of the integrated SARS spike protein gene. To quickly monitor the expression levels in small flask (T25 or T75) and in the three liter evaluation cultures, a lectin-based process (Gluvanthus Nivalis lectin) may be used to isolate SARS spike protein to a degree of purity that allows semi-quantitation and characterization of the protein in CHO supernatant. For full-length spike protein, it will be obtained from triton X-100 detergent extracted cells. Full-length Spike protein will be then captured on GNA lectin, followed by hydroxyapatite and SP chromatograph. Eluted protein is then characterized by: 1) polyacrylamide gel electrophoresis (PAGE) and Coomassie staining, 2) Immunoprobing with anti-SARS rabbit sera, 3) structural characterization using size exclusion chromatography (SEC), as well as mass spec analysis using MALDI-TOF.

Routes and methods of immunization of the vaccines of the invention are discussed in more detail in a section below. Examples 7 to 9 illustrate sample immunization protocols for the recombinant spike proteins.

Vaccine Testing

Prior to human administration, it is normal to test vaccines in animal models. A mouse model of SARS coronavirus infection is known (Subbarao et al. (2004) J Virol 78:3572-77), and other animals that may be used as models of infection and/or disease include ferrets and monkeys. Thus the invention provides a non-human animal that is infected by the SARS coronavirus, wherein the animal is preferably a ferret or a primate (e.g. a monkey or a macaque). The animal may be gnotobiotic. The animal is preferably not a cat (Felis domesticus). The animal may or may not display SARS disease symptoms e.g. ferrets (Mustela furo) show prominent pulmonary pathology after infection. See: Martina et al. (2003) Nature 425:915.

E. Polynucleotides Encoding the SARS Antigens of the Invention

The invention includes polynucleotides encoding for the SARS antigens of the invention. In addition, the invention includes polynucleotides which have been optimized for recombinant production (e.g. codon optimization) of the SARS antigens of the invention, including polynucleotides encoding for each of the SARS fusion constructs discussed above.

F. Viral Vector or Viral Particle Delivery of the SARS Antigens of the Invention

The antigens of the invention may be expressed in vivo or in vitro by polynucleotides encoding the antigens. Expression and delivery of the polynucleotides of the invention may be facilitated via viral vectors and/or viral particles.

Gene-based delivery systems derived from viruses, such as alphaviruses, are useful for the ex vivo and in vivo administration of heterologous genes, including one or more SARS genes, having therapeutic or prophylactic applications. These systems can also be used for the production of recombinant proteins derived from the SARS virus in cultured cells. Gene-based delivery systems of the invention include viral vectors (e.g., adenovirus vector, poxvirus vector, alphavirus vector) and non-viral nucleic acid vectors (e.g., DNA, RNA) encoding one or more SARS virus antigens. Polynucleotides encoding SARS virus antigen(s) are incorporated into the gene-based vaccines individually or in combination (e.g., as bicistronic constructs).

1. Alphavirus

Alphaviruses are members of Togaviridae family and share common structural and replicative properties. Sindbis virus (SIN) is the prototype virus for the molecular study of other alphaviruses, and together with Venezuelan equine encephalitis virus (VEE) and Semliki Forest virus (SFV), are the most widely utilized alphaviruses being developed into expression vectors for heterologous genes (Schlesinger and Dubensky (1999) Curr Opin. Biotechnol. 10:434-439; Schlesinger (2001) Expert Opin. Biol. Ther. 1:177-91).

Alphaviruses possess a relatively small single-stranded RNA genome of positive polarity, which is approximately 12 kb in length, capped and polyadenylated. The RNA interacts with viral capsid protein monomers to form nucleocapsids, which in turn, are surrounded by a host cell-derived lipid envelope from which two viral glycoproteins, E1 and E2, protrude forming “spike” trimers of heterodimeric subunits. Two open reading frames (ORFs) encode as polyproteins the enzymatic nonstructural replicase proteins (5′ ORF) and the virion structural proteins (3′ ORF). The structural polyprotein is translated from a highly abundant subgenomic mRNA, which is transcribed from a strong internal alphavirus promoter (Strauss and Strauss (1994) Microbiol. Rev. 58:491-562). Replication of the genome occurs exclusively within the host cell cytoplasm as RNA.

The most common alphavirus expression vectors have exploited both the positive-stranded nature and modular organization of the RNA genome. These vectors, termed “replicons” due to their property of self-amplification, permit insertion of heterologous sequences in place of the structural polyprotein genes, while maintaining the 5′- and 3′-end cis replication signals, the nonstructural replicase genes, and the subgenomic junction region promoter (Xiong et al. (1989) Science 243:1188-1191; Liljestrom (1991) Bio/Technology 9:1356-1361). Chimeric alphavirus vectors (and particles) from sequences derived from divergent virus families have also been described. (see, for example U.S. patent application Ser. No. 09/236,140; see also, U.S. Pat. Nos. 5,789,245, 5,842,723, 5,789,245, 5,842,723, and 6,015,694; as well as WO 95/07994, WO 97/38087 and WO 99/18226). Co-owned International Publication WO 02/099035, published Dec. 12, 2002 and incorporated by reference in its entirety herein, describes chimeric alphavirus molecules and modified alphavirus molecules having modified Biosafety Levels.

The absence of structural protein genes renders alphavirus replicon vectors defective, in that RNA amplification and high-level heterologous gene expression occurs within the target cell, but cell-to-cell spread of vector is not possible due to the inability to form progeny virions. Through the years, several synonymous terms have emerged that are used to describe alphavirus replicon particles. These terms include recombinant viral particle, recombinant alphavirus particle, alphavirus replicon particle and replicon particle. However, as used herein, these terms all refer to a virion-like unit containing an alphavirus-derived RNA vector replicon. Moreover, these terms may be referred to collectively as vectors, vector constructs or gene delivery vectors.

Packaging of replicon RNA into particles can be accomplished by introducing the replicon RNA into permissive cells (e.g., RNA or DNA transfection, or particle infection) that also contain one or more structural protein expression cassettes or “defective helper” constructs encoding the alphavirus structural proteins. These structural protein encoding constructs may themselves be introduced into the cells by transfection of either RNA or DNA, and most commonly retain the native alphavirus subgenomic promoter, as well as 5′- and 3′-end cis signals for co-amplification with the replicon, but are devoid of any replicase genes and the RNA packaging signal (Liljestrom (1991) Bio/Technology 9:1356-1361; Pushko et al. (1997) Virology 239:389-401; Polo et al. (1999) PNAS 96:4598-4603). Permanent cell lines that are stable transformed with constructs expressing the alphavirus structural proteins (e.g., packaging cell lines) offer a means to avoid transient transfection production methods (Polo et al. (1999) PNAS 96:4598-4603).

The present invention includes compositions and methods for the production of replication defective viral vector particles (e.g., alphavirus replicon particles) for use in the ex vivo and in vivo administration of heterologous genes encoding proteins having therapeutic or prophylactic application, including genes encoding for one or more SARS viral antigens.

In one aspect, the invention includes a method of producing replication defective viral vector particles (e.g., alphavirus replicon particles) comprising the steps of introducing at least one nucleic acid molecule comprising a viral vector (e.g., alphavirus replicon RNA) into immortalized cells of the present invention, under conditions that allow for complementation of the viral vector (e.g., alphavirus replicon RNA) and production of viral vector particles (e.g., alphavirus replicon particles), and isolating the viral vector particles from the cells or cell culture supernatants. In certain embodiments, the immortalized cells are grown in suspension, for example PERC.6 cells. In other embodiments, the methods are performed in large-scale volumes, for example, liter volumes or greater, such as for example in roller bottles, large flasks, Nunc Cell Factories, Corning Cell Cubes, fermentation vessels, etc).

In certain embodiments, the viral vector is an alphavirus replicon RNA that requires complementation by providing one or more alphavirus structural proteins in trans, within the immortalized cell. In such instances, the methods of complementation to produce alphavirus replicon particles may involve the introduction of one or more nucleic acids (e.g., RNA, DNA) encoding said alphavirus structural protein(s) (e.g., capsid and/or envelope glycoproteins) into the immortalized cells, either transiently or stably, and either concurrent with or prior to the introduction of the alphavirus replicon RNA. In certain embodiments, the alphavirus replicon RNA is introduced into the cell by transfection an in vitro transcribed RNA. In other embodiments, the alphavirus replicon RNA is introduced into the cell by transfection of a DNA (e.g., ELVIS), which is capable of transcribing within the cell, the replicon RNA. In yet other embodiments, the alphavirus replicon RNA is introduced into the cell by infection with a seed stock of alphavirus replicon particles. In certain embodiments, the nucleic acids encoding said alphavirus structural protein(s) are defective helper RNA or are DNA that can transcribe within the cell defective helper RNAs.

As discussed herein, “alphavirus RNA replicon vector”, “RNA replicon vector”, “replicon vector” or “replicon” refers to an RNA molecule that is capable of directing its own amplification or self-replication in vivo, within a target cell. To direct its own amplification, the RNA molecule should encode the polymerase(s) necessary to catalyze RNA amplification (e.g., alphavirus nonstructural proteins nsP1, nsP2, nsP3, nsP4) and also contain cis RNA sequences required for replication which are recognized and utilized by the encoded polymerase(s). An alphavirus RNA vector replicon should contain the following ordered elements: 5′ viral or cellular sequences required for nonstructural protein-mediated amplification (may also be referred to as 5′ CSE, or 5′ cis replication sequence, or 5′ viral sequences required in cis for replication, or 5′ sequence which is capable of initiating transcription of an alphavirus), sequences which, when expressed, code for biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), and 3′ viral or cellular sequences required for nonstructural protein-mediated amplification (may also be referred as 3′ CSE, or 3′ viral sequences required in cis for replication, or an alphavirus RNA polymerase recognition sequence). The alphavirus RNA vector replicon also should contain a means to express one or more heterologous sequence(s), such as for example, an IRES or a viral (e.g., alphaviral) subgenomic promoter (e.g., junction region promoter) which may, in certain embodiments, be modified in order to increase or reduce viral transcription of the subgenomic fragment, or to decrease homology with defective helper or structural protein expression cassettes, and one or more heterologous sequence(s) to be expressed. Preferably the heterologous sequence(s) comprises a protein-encoding gene, which is the 3′ proximal gene within the vector replicon. And preferably the replicon further comprises a polyadenylate tract.

As discussed herein, “recombinant Alphavirus Particle”, “alphavirus replicon particle” and “replicon particle” refers to a virion-like unit containing an alphavirus RNA vector replicon. Generally, the recombinant alphavirus particle comprises one or more alphavirus structural proteins, a lipid envelope and an RNA vector replicon. Preferably, the recombinant alphavirus particle contains a nucleocapsid structure that is contained within a host cell-derived lipid bilayer, such as a plasma membrane, in which one or more alphaviral envelope glycoproteins (e.g., E2, E1) are embedded. The particle may also contain other components (e.g., targeting elements such as biotin, other viral structural proteins or portions thereof, hybrid envelopes, or other receptor binding ligands), which direct the tropism of the particle from which the alphavirus was derived. Generally the interaction between alphavirus RNA and structural protein(s) necessary to efficiently form a replicon particle or nucleocapsid may be an RNA-protein interaction between a capsid protein and a packaging signal or packaging sequence contained within the RNA.

“Alphavirus packaging cell line” refers to a cell which contains one or more alphavirus structural protein expression cassettes and which produces recombinant alphavirus particles (replicon particles) after introduction of an alphavirus RNA vector replicon, eukaryotic layered vector initiation system, or recombinant alphavirus particle. The parental cell may be of mammalian or non-mammalian origin. Within preferred embodiments, the packaging cell line is stably transformed with the structural protein expression cassette(s).

“Defective helper RNA” refers to an RNA molecule that is capable of being amplified and expressing one or more alphavirus structural proteins within a eukaryotic cell, when that cell also contains functional alphavirus nonstructural “replicase” proteins. The alphavirus nonstructural proteins may be expressed within the cell by an alphavirus RNA replicon vector or other means. To permit amplification and structural protein expression, mediated by alphavirus nonstructural proteins, the defective helper RNA molecule should contain 5′-end and 3′-end RNA sequences required for amplification, which are recognized and utilized by the nonstructural proteins, as well as a means to express one or more alphavirus structural proteins. Thus, an alphavirus defective helper RNA should contain the following ordered elements: 5′ viral or cellular sequences required for RNA amplification by alphavirus nonstructural proteins (also referred to elsewhere as 5′ CSE, or 5′ cis replication sequence, or 5′ viral sequences required in cis for replication, or 5′ sequence which is capable of initiating transcription of an alphavirus), a means to express one or more alphavirus structural proteins, gene sequence(s) which, when expressed, codes for one or more alphavirus structural proteins (e.g., C, E2, E1), 3′ viral or cellular sequences required for amplification by alphavirus nonstructural proteins (also referred to as 3′ CSE, or 3′ viral sequences required in cis for replication, or an alphavirus RNA polymerase recognition sequence), and a preferably a polyadenylate tract. Generally, the defective helper RNA should not itself encode or express in their entirety all four alphavirus nonstructural proteins (nsP1, nsP2, nsP3, nsP4), but may encode or express a subset of these proteins or portions thereof, or contain sequence(s) derived from one or more nonstructural protein genes, but which by the nature of their inclusion in the defective helper do not express nonstructural protein(s) or portions thereof. As a means to express alphavirus structural protein(s), the defective helper RNA may contain a viral (e.g., alphaviral) subgenomic promoter which may, in certain embodiments, be modified to modulate transcription of the subgenomic fragment, or to decrease homology with replicon RNA, or alternatively some other means to effect expression of the alphavirus structural protein (e.g., internal ribosome entry site, ribosomal readthrough element). Preferably an alphavirus structural protein gene is the 3′ proximal gene within the defective helper. In addition, it is also preferable that the defective helper RNA does not contain sequences that facilitate RNA-protein interactions with alphavirus structural protein(s) and packaging into nucleocapsids, virion-like particles or alphavirus replicon particles. A defective helper RNA is one specific embodiment of an alphavirus structural protein expression cassette.

Alphavirus for use in the invention may be grown in any one of the cell lines discussed above as suitable for the SARS virus.

Alphavirus replicon particles may be produced according to the present invention by using the above cell lines (e.g., immortalized cell lines) and a variety of published and accepted alphavirus vector methodologies. Such methodologies include, for example, transient packaging approaches, such as the co-transfection of in vitro transcribed replicon and defective helper RNA(s) (Liljestrom, Bio/Technology 9:1356-1361, 1991; Bredenbeek et al., J. Virol. 67:6439-6446, 1993; Frolov et al., J. Virol. 71:2819-2829, 1997; Pushko et al., Virology 239:389-401, 1997; U.S. Pat. Nos. 5,789,245 and 5,842,723) or co-transfection of plasmid DNA-based replicon and defective helper construct(s) (Dubensky et al., J. Virol. 70:508-519, 1996), as well as introduction of alphavirus structural protein expression cassettes (e.g., DNA-based defective helper) into immortalized cell lines of the present invention to create stable packaging cell lines (PCL) (Polo et al., PNAS 96:4598-4603, 1999; U.S. Pat. Nos. 5,789,245, 5,842,723, 6,015,694; WO 97/38087, WO 99/18226, WO 00/61772, and WO 00/39318). Stable packaging cell lines may then be utilized for alphavirus replicon particle production. The PCL may be transfected with in vitro transcribed alphavirus replicon RNA, transfected with a plasmid DNA-based replicon (e.g., ELVIS vector), or infected with a seed stock of alphavirus replicon particles, and then incubated under conditions and for a time sufficient to produce progeny alphavirus replicon particles in the culture supernatant. In addition, progeny replicon particles can subsequently be passaged in additional cultures of naive PCL by infection, resulting in further expansion and commercial scale preparations. Importantly, by using defective helper RNA or stable PCL based on the “split” structural gene configuration, these replicon particle stocks may be produced free from detectable contaminating RCV.

Following harvest, crude culture supernatants containing the chimeric alphavirus replicon particles may be clarified by passing the harvest through a filter (e.g., 0.2 uM, 0.45 uM, 0.65 uM, 0.8 uM pore size). Optionally, the crude supernatants may be subjected to low speed centrifugation prior to filtration to remove large cell debris. Within one embodiment, an endonuclease (e.g., Benzonase, Sigma #E8263) is added to the preparation of alphavirus replicon particles before or after a chromatographic purification step to digest exogenous nucleic acid. Further, the preparation may be concentrated prior to purification using one of any widely known methods (e.g., tangential flow filtration). Crude or clarified alphavirus replicon particles may be concentrated and purified by chromatographic techniques (e.g., ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography), such as those described in WO01/92552, incorporated by reference in its entirety herein. Two or more such purification methods may be performed sequentially.

Example of Alphavirus Replicon Particles Encoding SARS Virus Spike (S) Antigen

The invention includes compositions and methods for the production of replication defective viral vector particles (e.g., alphavirus replicon particles) for use in the ex vivo and in vivo administration of heterologous genes encoding proteins having therapeutic or prophylactic application, including genes encoding for one or more SARS viral antigens.

The following example illustrates a method of preparing alphavirus replicon particles encoding SARS virus spike (s) antigen.

The SARS virus spike gene can be incorporated into alphavirus replicon particles derived from a variety of alphavirus, such as Sindbis virus, Semliki Forest virus (U.S. Pat. No. 5,739,026), Venezuelan equine encephalitis virus (U.S. Pat. No. 6,531,135), and replicon particle chimeras derived from more than one alphavirus (U.S. Pat. No. 6,376,236, WO 02/99035). In addition, the SARS virus spike gene can be incorporated in its entirety (encoding full-length spike protein) or in a modified form that includes, for example, sequence deletions or truncations, such that the encoded a spike protein is of less than full-length (e.g., C-terminal truncation of one or more (e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 etc.) amino acids, deleted of transmembrane region and cytoplasmic tail).

For example, the spike gene may be cloned as a full-length gene into the VCR-chim2.1 vector (WO 02/99035) by standard RT-PCR conditions or by standard subcloning from one of the other plasmids described herein, using commercially available restriction endonucleases. For the reverse transcription step in standard RT-PCR, the Superscript pre-amplification kit (Invitrogen™) and the primer SEQ ID NO: 7325 (sp-RT-R) are used:

For the amplification step, the cDNA polymerase advantage kit (Clonetech) and two primers Sp-F-BbvCI (SEQ ID NO: 7326) and Sp-R-NotI (SEQ ID NO: 7327) are used:

The forward primer is designed to contain the ccacc sequence (Kozak, 1991 JBC 19867-70) in front of the ATG codon to optimize translation efficiency of the spike gene. Also, the forward primer contains the BbvCI restriction site and the reverse primer contains the NotI restriction site for subsequent cloning of the PCR amplified gene.

The PCR product is purified using the QIAquick Nucleotide Removal kit (QIAgen), digested with BbvCI and NotI, gel purified with QIAquick Gel Extraction kit (QIAgen), and ligated to plasmid VCR-Chim2.1 pre-digested with the same enzymes. Clones containing the SARS spike sequence are verified by sequencing and the new construct is called VCR-Chim2.1-SARSspike.

To generate VEErep/SINenv-SARSspike replicon particles the plasmids VCR-Chim2.1-SARSspike, VCR-DH-Scap (WO 02/99035), and VCR-DH-Sglyd1160 (WO 02/99035) are linearized with the restriction enzyme PmeI and used for in vitro transcription as described previously (Polo et al. 1999, PNAS 96: 4598-603; WO02/99035). The transcripts are co-transfected into BHK cells as previously described (Polo et al., 1999, ibid.; WO02/99035). The transfected cells are incubated at 34° C., the supernatants collected at 20 and 30 hrs post-electroporation, clarified by centrifugation, and purified by chromatography as previously described (WO 01/92552).

Expression of the SARS spike protein from the replicon particle vector is verified by infecting BHK cells overnight with purified VEErep/SINenv-SARSspike or VEErep/SINenv-GFP (WO 02/99035) replicon particles. In addition, BHK cells also were transfected in parallel with in vitro transcribed VCR-Chim2.1-SARSspike replicon RNA. At 16 hrs post-infection and transfection cells are lysed and a sample of the lysate analyzed by western blot using an antibody that recognizes SARS virus spike protein. The proteins on the gel are stained or transferred to a membrane for Western blot analysis with sera from convalescent patients or alternatively murine or rabbit antisera generated against SARS virus. VEErep/SINenv-SARSspike replicon particles are administered to the vaccine recipient (e.g., rodent, non-human primate, human) as described elsewhere in the present invention.

FIG. 67 shows data from western blot analysis performed under non-reducing conditions, using a SARS virus specific rabbit polyclonal antisera. The western data demonstrate that not only is SARS spike protein expressed in cells infected with alphavirus replicon particles or transfected with replicon RNA, but the predominant form of spike is that of a homotrimer (FIG. 67A). Similar homotrimeric association of the spike protein was observed in western blots of SARS virions purified from SARS virus infected VERO cell supernatants, and this homotrimer is heat labile, as indicated by the dissociation into monomeric forms at 80° C. and 100° C. (FIG. 67B).

To further characterize SARS Spike protein expression and processing following expression from alphavirus replicon vectors, BHK-21 cells were infected with alphavirus replicon particles expressing the full-length Spike. At 6 hr post-infection with an MOI of 5, infected cells were labeled for 1 hr with L-[³⁵S]methionine/cysteine and chased for the indicated time. The [³⁵S]-labeled spike protein was immunoprecipitated by anti-SARS rabbit serum and digested with Endo-H. Both digested and undigested proteins were analysed by 4% polyacrylamide-SDS PAGE under reducing conditions. As shown in FIG. 55, the full-length spike protein is synthesized as an Endo-H sensitive high mannose glycoprotein (gp170, an ER form) that undergoes modification to an Endo-H resistant glycoprotein with complex oligosaccharides (gp180, a Golgi form). The conversion of gp170 into the gp180 form takes place within 2 hr.

Alphavirus replicon particles expressing one or more SARS proteins (e.g., VEErep/SINenv-SARSspike replicon particles) are administered to the vaccine recipient in order to induce a SARS specific immune response (e.g., rodent, ferret, non-human primate, human) as described elsewhere in the present invention. Immunization may be performed through a variety of routes, including for example, intramuscular, subcutaneous, intradermal, and intranasal. In additon, the alphavirus replicon particles may be used alone or in combination (e.g., “prime-boost”) with other vaccine approaches of the present invention, or alternatively the alphavirus replicon particles may co-express antigen from other respiratory pathogens or be co-administered in combination with alphavirus replicon particles expressing antigens from other respiratory pathogens (e.g., influenza virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus). For example, the induction of anti-spike protein antibodies in animals immunized IM with VEErep/SINenv-SARSspike replicon particles was demonstrated in mice (FIG. 68). These mouse studies also included addtional vaccine groups for comparison, including the inactivated SARS virus and recombinant truncated spike protein vaccines describe elsewhere herein, as well as plasmid DNA used as a prime, followed by alphavirus replicon particles as a boost. The data clearly show very potent immune responses for all vaccine groups, including the alphavirus replicon particle group. It should be noted that the level of antibody induced by the inactivated SARS virus vaccine used in these experiments has been shown to be protective in a SARS virus animal challenge model.

Similarly, genes encoding other SARS virus antigens (e.g., nucleocapsid protein, membrane glycoprotein) are cloned into alphavirus replicon vectors, either individually or in combination, to generate alphavirus replicon particles according to the teachings of the present invention and using standard molecular biology techniques.

Example of Alphavirus-Based Plasmid DNA Expressing SARS Virus Spike (S)

The invention includes preparation of plasmid DNA expressing a SARS virus antigen for prophylactic or therapeutic immunization against SARS virus infection. In one embodiment, the SARS viral antigen is a spike (S) protein. In one embodiment, the plasmid DNA is alphavirus-based.

The following example illustrates one method for preparing an alphavirus-based plasmid DNA expressing SARS virus spike (S).

SARS spike gene can be delivered using any of the alphavirus-based plasmid DNA replicons such as ELVS (Dubensky et al, 1996 J Virol. 70: 508-19), SINCP (WO 01/81609), or VCP (PCT WO 02/99035).

For example, the SARS spike gene is cloned into SINCP using the standard RT-PCR techniques. The oligo Sp-RT-R is used for the reverse transcription step with the Superscript pre-amplification kit (Invitrogen). For the amplification step, the cDNA polymerase advantage kit (Clonetech) with the Sp-R-NotI and Sp-F-XhoI (SEQ ID NO: 7328) primers is used.

The Sp-F-XhoI primer was designed to contain the ccacc sequence in front of the ATG codon to optimize translation efficiency (Kozak 1991, ibid) of the spike gene. Also, the primer contains the XhoI restriction site for the subsequent cloning of the PCR amplified gene.

The PCR product is purified using the QIAquick Nucleotide removal kit, digested with XhoI and NotI, gel purified with QIAquick Gel Extraction kit, and ligated to plasmid SINCP pre-digested with the same enzymes. Clones containing the SARS spike sequence are verified by sequencing and the new construct is called SINCP-SARSspike.

Expression of the SARS spike gene is verified by transient transfection of BHK cells with 2 μg of either plasmid DNA SINCP-SARSspike or SINCP pre-incubated for 5 minutes with 5 μl of TransIT Polyamine reagent (Mirrus) in low serum medium Optimem (Life Technologies). At 48 hrs pos-transfection cells are lysed and a sample of the lysate is run on 8% SDS-PAGE. The proteins on the gel are either stained or transferred to a membrane for Western blot analysis with sera from convalescent patients, or alternatively with sera from mouse or rabbits.

SINCP-SARSspike plasmid replicon is administered to the vaccine recipient (e.g., rodent, non-human primate, human) as a formulated or unformulated plasmid vaccine, alone or in combination (e.g., “prime-boost”) with other vaccines of the present invention, as described elsewhere herein.

Similarly, genes encoding other SARS virus antigens (e.g., nucleocapsid protein, membrane glycoprotein) are cloned into alphavirus plasmid replicon vectors.

2. Plasmid Expression Vectors

Example of Plasmid DNA Expressing SARS Virus Spike (S)

The following example illustrates a method for preparing plasmid DNA expressing SARS virus spike (s).

The SARS virus spike antigen also may be delivered using other plasmid DNA expression vectors (sometimes referred to as “conventional” DNA vaccines), based on a polymerase II promoter, such as, for example, a CMV promoter. A DNA vaccine of the spike antigen gene induces an antibody response in mice (Zhao et al. (2004) Acta Biochim et Biophysica Sinica 36:37-41), and has been found to induce viral neutralization and protective immunity in mice (Yang et al. (2004) Nature 428:561-564), particularly when truncated at the C-terminus.

For example, the SARS spike gene is cloned into pCMVKm2 (Zur Megede et al., J. Virol., 74:2628-2635, 2000; SEQ ID NO: 9923) using standard RT-PCR techniques. The oligo Sp-RT-R is used for the reverse transcription step with the Superscript pre-amplification kit (Invitrogen). For the amplification step, the cDNA polymerase advantage kit (Clonetech) is used with primers Sp-F-EcoRI (SEQ ID NO: 7329) and Sp-R-XbaI (SEQ ID NO: 7330).

The forward primer was designed to contain the CCACC (SEQ ID NO: 7331) sequence in front of the ATG codon to optimize translation efficiency (Kozak 1991, ibid.) of the spike gene. Also, the forward primer contains the EcoRI restriction site and the reverse primer contains the XbaI restriction site for the subsequent cloning of the PCR amplified gene.

The PCR product is purified using the QIAquick Nucleotide Removal kit, digested with XhoI and NotI, gel purified with QIAquick Gel Extraction kit, and ligated to plasmid pCMVKm2 pre-digested with the same enzymes. Clones containing the SARS spike sequence are verified by sequencing and the new construct is called pCMVKm2-SARSspike.

Expression of the SARS spike gene is verified by transient transfection of BHK or 293 cells with 2 μg of either plasmid DNA pCMVKm2-SARSspike or pCMVKm2 pre-incubated for 5 minutes with 5 μl of TransIT Polyamine reagent (Mirrus) in low serum medium Optimem (Life Technologies). At 48 hrs pos-transfection cells are lysed and a sample of the lysate is run on 8% SDS-PAGE. The proteins on the gel are either stained or transferred to a membrane for Western blot analysis with sera from convalescent patients, or alternatively using mouse or rabbit antisera.

Plasmid pCMVKm2-SARSspike is administered to the vaccine recipient (e.g., rodent, non-human primate, human) as a formulated or unformulated plasmid vaccine, as described elsewhere in the present invention.

Similarly, genes encoding other SARS virus antigens (e.g., nucleocapsid protein, membrane glycoprotein) are cloned into plasmid expression vectors

3. Virus-Like Particles Comprising SARS Antigens

The SARS viral antigens of the invention may be formulated into Virus Like Particles (“VLPs”). The invention thus includes virus-like particles (or VLPs) comprising one or more SARS viral antigens. Preferably, the VLPs comprise one or more SARS viral antigens selected from the group consisting of Spike (S), nucleocapsid (N), membrane (M) and envelope (E). Preferably, the VLPs comprise at least M and E.

The VLPs of the invention comprise at least one particle-forming polypeptide. Said particle-forming polypeptide is preferably selected from a Coronavirus structural protein. In one embodiment, the particle-forming polypeptide is selected from one or more SARS viral antigens. In another embodiment, the particle-forming polypeptide is selected from the structural protein of a non-SARS Coronavirus, such as, for example, Mouse Hepatitis Virus.

VLPs can be formed when viral structural proteins are expressed in eukaryotic or prokaryotic expression systems. Upon expression, the structural proteins self-assemble to form particles. Alternatively, viral structural proteins may be isolated from whole virus and formulated with phospholipids. Such viral structural proteins are referred to herein as “particle-forming polypeptides”. VLPs are not infectious because no viral genome is present, however, these non-replicating, virus capsids mimic the structure of native virions.

Due to their structure, VLPs can display a large number of antigenic sites on their surface (similar to a native virus). VLPs offer an advantage to live or attenuated vaccines in that they are much safer to both produce and administer, since they are not infectious. VLPs have been shown to induce both neutralizing antibodies as well as T-cell responses and can be presented by both class I and II MHC pathways.

Previous work creating VLPs from coronavirus indicates that E and M proteins along may be sufficient for coronavirus VLP formation. See Fischer et al., J. Virol. (1998) 72:7885-7894 and Vennema et al. EMBO J. (1996) 15:2020-2028.

Chimeric VLPs comprising particle-forming polypeptides or portions thereof from non-SARS Coronaviruses are also included in the invention. Such particle-forming polypeptides may comprise a full length polypeptide from a non-SARS Coronavirus. Alternatively, a particle-forming fragment may be used.

In one embodiment, a fragment of a non-SARS particle-forming polypeptide and a fragment of a SARS viral antigen are fused together. For instance, such chimeric polypeptides may comprise the the endodomain and transmembrane domain of a non-SARS particle-forming polypeptide and the ectodomain of a SARS viral antigen. In one example, the VLPs of the invention comprise a chimeric spike protein comprising an endodomain and transmembrane domain of the spike protein of Mouse Hepatitis Virus (MHV) and the chimeric spike protein further comprises the ectodomain of the SARS spike protein. Such VLPs may further comprise Coronavirus M and E proteins. Said M and E proteins may be selected from any coronavirus, including Mouse Hepatitis Virus (MHV) or SARS. Sample sequences of S, M and E proteins of MHV are included in the figures, supra.

Chimeric spike proteins derived from the ectodomain of feline infectious peritonitis virus (FIPV) spike protein fused to the endo and transmembrane domains of MHV spike protein have been previously disclosed. See WO 98/49195 and WO 02/092827. In these chimeric VLP structures, the capsid structure of the VLPs is formed by the M and E protein of MHV. The chimeric spike protein provides for the surface exposure of the ectodomain of the FIPV spike protein.

As used herein, the term “virus-like particle” or “VLP” refers to a non-replicating, empty virus shell. VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an approrpirate expression system. Alternatively, viral structural proteins may be isolated from whole virus and formulated with phospholipids. Methods for producing particular VLPs are known in the art and discussed more fully below. The presence of VLPs in a composition can be detected using conventional techniques known in the art, such as by electron microscopy, x-ray crystallography, and the like. See, e.g., Baker et al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol. (1994) 68:4503-4505. For example, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.

The phrase “particle-forming polypeptide” includes a full-length or near full-length viral protein, as well as a fragment thereof, or a viral protein with internal deletion, which has the ability to form VLPs under conditions that favor VLP formation. Accordingly, the polypeptide may comprise the full-length sequence, fragments, truncated and partial sequences, as well as analogs and precursor forms of the reference molecule. The term therefore includes deletions, additions and substitutions to the sequence, so long as the polypeptide retains the ability to form a VLP. Thus, the term includes natural variations of the specified polypeptide since variations in coat proteins often occur between viral isolates. The term also includes deletions, addition and substitutions that do not naturally occur in the reference protein, so long as the protein retains the ability to form a VLP.

Preferred substitutions are those which are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic: aspartate and glutamate; (2) basic: lysine, arginine, and histidine; (3) non-polar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar: glycine, asparagine, glutamine, cystine, serine, theronine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an asparate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein, are therefore within the definition of the reference polypeptide.

The VLPs of the invention can be formed from any viral protein, particle-forming polypeptide derived from the viral protein, or combination of viral proteins or fragments thereof, that have the capability of forming particles under appropriate conditions. The requirements for the particle-forming viral proteins are that if the particle is formed in the cytoplasm of the host cell, the protein must be sufficiently stable in the host cell in which it is expressed such that formation of virus-like structures will result, and that the polypeptide will automatically assemble into a virus-like structure in the cell of the recombinant expression system used. If the protein is secreted into culture media, conditions can be adjusted such that VLPs will form. Furthermore, the particle-forming protein should not be cytotoxic in the expression host and should not be able to replicate in the host in which the VLP will be used.

Preferred particle-forming polypeptides include coronavirus M and E proteins, preferably SARS M and E proteins.

Methods and suitable conditions for forming particles from a wide variety of viral proteins are known in the art. VLPs have been produced, for example from proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p1). VLPs are discussed further in WO 03/024480, WO 03/024481, and Niikura et al., Virology (2002) 293:273-280; Lenz et al., J. Immunology (2001) 5246-5355; Pinto, et al., J. Infectious Diseases (2003) 188:327-338; and Gerber et al., J. Virology (2001) 75(10):4752-4760.

As explained above, VLPs can spontaneously form when the particle-forming polypeptide of interest is recombinantly expressed in an appropriate host cell. Thus, the VLPs for use in the present invention may be prepared using recombinant techniques, well known in the art. In this regard, genes encoding the particle-forming polypeptide in question can be isolated from DNA libraries or directly from cells and tissues containing the same, using known techniques. The genes encoding the particle-forming polypeptides can also be produced synthetically, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed (e.g. human codons for human DNA vaccines). The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See., e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311.

Once the coding sequences for the desired particle-forming polypeptides have been isolated or synthesized, they can be cloned into any suitable vector or replicon for expression. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. See, generally, Sambrook et al. The vector is then used to transform an appropriate host cell. Suitable expression systems include, but are not limited to, bacterial, mammalian, bacuolvirus/insect, vaccinia, Semliki Forest virus (SFV), yeast, and Xenopus expression systems, well known in the art.

A number of cell lines suitable for use as host cells for producing the VLPs of the invention are known in the art. Suitable mammalian cell lines include, but are not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Mammalian sources of cells include, but are not limited to, human or non-human primate (e.g., MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), HUH, human embryonic kidney cells (293 cells, typically transformed by sheared adenovirus type 5 DNA), VERO cells from monkey kidneys (including, for example COS7 cells), horse, cow (e.g., MDBK cells), sheep, dog (e.g., MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 as described in WO 97/37001), cat, and rodent (e.g., hamster cells such as BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and may be obtained from a wide variety of developmental stages, including for example, adult, neonatal, fetal, and embryo.

Bacterial hosts suitable for production of VLPs of the invention include E. coli, Bacillus subtilis, and Streptoccocus spp. Yeast hosts suitable for production of VLPs of the invention include Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells suitable for production of VLPs of the invention (i.e., via baculovirus expression vectors) include Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodptera frugiperda, and Trichoplusia ni.

Viral vectors can be used for the production of particles in eukaryotic cells, such as those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. Additional, vaccinia based infection/transfection systems, such as those as described in Tomei et al., J. Virol (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-1113, can also be used to generate the VLPs of the invention. In this system, cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translation machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products.

Depending on the expression system and host selected, the VLPs are produced by growing host cells transformed by an expression vector under conditions whereby the particle-forming polypeptide is expressed and VLPs can be formed. The selection of the appropriate growth conditions is within the skill of the art. If the VLPs are formed intracellularly, the cells are then disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the VLPs substantially intact. Such methods are known the those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (E. L. V. Harris and S. Angal, Eds., 1990).

The particles are then isolated using methods that preserve the integrity thereof, such as by gradient centrifugation, e.g., cesium chloride (CsCl) and sucrose gradients, and the like (see, e.g., Kirnbauer et al., J. Virol. (1993) 67:6929-6936), ion exchange chromatography (including anion exchange chromatography such as DMAE and TMAE), hydroxyapatitie chromatography (see WO 00/09671), hydrophobic interaction chromatography, gel filtration chromatography and other filtration methods such as nanometric filtration and ultrafiltration. Preferably at least one anion exchange step is performed during purification, and more preferably at least two anion exchange steps are used.

VLP formulations of the invention may be further processed by methods known in the art to disassemble the VLPs into smaller, protein containing moieties using a high concentration of reducing agent, followed by reassembly of the VLPs by either removal of the reducing agent or by addition of excess oxidant. The resulting reassembled VLPs may have improved homogeneity, stability and immunogenic properties. In addition, further therapeutic or prophylactic agents may be formulated into the VLPs upon reassembly. See McCarthy et al., J. Virology (1998) 72(1):32-41. See also WO 99/13056 and WO 01/42780. Reducing agents suitable for use in VLP disassembly include sulfhydryl reducing agents (such as glutathion, beta mercaptoethanol, dithiothreitol, dithioerythritol, cysteine, hydrogen sulfide and mixtures thereof) preferably contained in moderate to low ionic strength buffers. Sufficient exposure time of the VLPs to the reducing agent will be required to achieve a suitable amount of VLP disassembly.

Adjuvants may be added to the VLPs of the invention to enhance the immunogenicity of the SARS viral antigens. Antigens suitable for use with VLPs include those described, supra. For example, the VLPs of the invention may be adsorbed onto an aluminum adjuvant.

The VLPs of the invention may formulated to enhance their stability. Additional components which may enhance the stability of a VLP formulation include salts, buffers, non-ionic surfactants and other stabilizers such as polymeric polyanion stabilizers. See WO 00/45841.

The ionic strength of a solution comprising VLP particles may be maintained by the presence of salts. Almost any salt which can contribute to the control of the ionic strength may be used. Preferred salts which can be used to adjust ionic strength include physiologically acceptable salts such as NaCl, KCl, Na₂SO₄, (NH₄)₂SO₄, sodium phosphate and sodium citrate. Preferably, the salt component is present in concentrations of from about 0.10 M to 1 M. Very high concentrations are not preferred due to the practical limitations of parenteral injection of high salt concentrations. Instead, more moderate salt concentrations, such as more physiological concentrations of about 0.15M to about 0.5M with 0.15M-0.32M NaCl are preferred.

Buffers may also be used to enhance the stability of the VLP formulations of the invention. Preferably, the buffer optimizes the VLP stability while maintaining the pH range so that the vaccine formulation will not be irritating to the recipient. Buffers preferably maintain the pH of the vaccine formulation within a range of p/H 5.5-7.0, more preferably 6.0-6.5. Buffers suitable for vaccine formulations are known in the art and include, for example, histidine and imidazole. Preferably, the concentration of the buffer will range from about 2 mM to about 100 mM, more preferably 5 mM to about 20 mM. Phosphate containing buffers are generally not preferred when the VLP is adsorbed or otherwise formulated with an aluminum compound.

Non-ionic surfactants may be used to enchance the stability of the VLP formulations of the invention. Surfactants suitable for use in vaccine formulations are known in the art and include, for example, polyoxyethylene sorbital fatty acid esters (Polysorbates) such as Polysorbate 80 (e.g., TWEEN 80), Polysorbate 20 (e.g., TWEEN 20), polyoxyethylene alkyl ethers (e.g., Brij 35, Brij 58), as well as others, including Triton X-100, Triton X-114, NP-40, Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121). The surfactant is preferably present in a concentration of from about 0.0005% to about 0.5% (wt/vol).

Polymeric polyanion stabilizers may also be used to enchance the stability of the VLP formulations of the invention. Suitable polymeric polyanionic stabilizers for use in the invention comprise either a single long chain or multiple cross linked chains; either type possessing multiple negative charges along the chains when in solution. Examples of suitable polyanionic polymers include proteins, polyanions, peptides and polynucelic acids. Specific examples include carboxymethyl cellulose, heparin, polyamino acids (such as poly(Glu), poly(Asp), and Poly (Glu, Phe), oxidized glutathione, polynuceltodies, RNA, DNA and serum albumins. The concentration of the polmeric polyanion stabilizers is preferably from about 0.01% to about 0.5%, particularly about 0.05-0.1% (by weight).

G. Passive Immunization via Antibodies to the SARS Antigens of the Invention

The invention includes antibodies specific to the SARS antigens of the invention and methods of treatment or prevention of SARS virus related disease by administrating an effective amount of SARS antibodies to a mammalian subject. Antibodies specific the SARS antigens can be produced by one skilled in the art. Preferably, the antibodies are specific to the spike (S) protein of the SARS virus. Potent neutralization of the SARS coronavirus using a human monoclonal anti-spike antibody has been reported (Sui et al. (2004) PNAS USA 101:2536-2541). A IgG1 form of the monoclonal antibody showed a higher affinity (1.59 nM) than a scFv form (32.3 nM).

The antibodies of the invention are specific and selective to SARS antigens.

In one embodiment, the antibodies of the invention are generated by administering a SARS antigen to an animal. The method may also include isolating the antibodies from the animal.

The antibodies of the invention may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, or may be hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies (Fab′)₂ fragments, F(ab) fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragments or constructs, minibodies, or functional fragments thereof which bind to the antigen in question.

Antibodies are produced using techniques well known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745. For example, polyclonal antibodies are generated by immunizing a suitable animal, such as a mouse, rat, rabbit, sheep, or goat, with an antigen of interest. In order to enhance immunogenicity, the antigen can be linked to a carrier prior to immunization. Such carriers are well known to those of ordinary skill in the art. Immunization is generally performed by mixing or emulsifying the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). The animal is generally boosted 2-6 weeks later with one or more injections of the antigen in saline, preferably using Freund's incomplete adjuvant. Antibodies may also be generated by in vitro immunization, using methods known in the art. Polyclonal antiserum is then obtained from the immunized animal.

Monoclonal antibodies are generally prepared using the method of Kohler & Milstein (1975) Nature 256:495-497, or a modification thereof. Typically, a mouse or rat is immunized as described above. Rabbits may also be used. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen. B-cells, expressing membrane-bound immunoglobulin specific for the antigen, will bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice).

Humanized and chimeric antibodies are also useful in the invention. Hybrid (chimeric) antibody molecules are generally discussed in Winter et al. (1991) Nature 349: 293-299 and U.S. Pat. No. 4,816,567. Humanized antibody molecules are generally discussed in Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994). One approach to engineering a humanized antibody involves cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse antibody gene and the constant-region exons from a human antibody gene to create a mouse-human chimera, a humanized antibody. See generally, Kuby, “Immunology, 3^(rd) Edition”, W.H. Freeman and Company, New York (1998) at page 136.

Antibody fragments which retain the ability to recognize a SARS antigen are also included within the scope of the invention. A number of antibody fragments are known in the art which comprise antigen-binding sites capable of exhibiting immunological binding properties of an intact antibody molecule. For example, functional antibody fragments can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g., pepsin, to produce F(ab′)₂ fragments. These fragments will contain two antigen binding sites, but lack a portion of the constant region from each of the heavy chains. Similarly, if desired, Fab fragments, comprising a single antigen binding site, can be produced, e.g., by digestion of polyclonal or monclonal antibodies with papain. Functional fragments, includnig only the variable regions of the heavy and light chains, can also be produced, using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as F_(v). See, e.g., Inbar et al. (1972) Proc. Nat. Acad. Sci USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single-chain Fv (“sFv” or scFv”) polypeptide is a covalently linked V_(H)-V_(L) heterodimer which is expressed from a gene fusion including V_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883. A number of methods have been described to discern and develop chemical strucutres (linkers) for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405; and 4,946,778. The sFv molecules may be produced using methods described in thea rt. See, e.g., Huston et al. (1988) Proc. Nat. Acad. Sci USA 85:5879-5338; U.S. Pat. Nos. 5,091,513; 5,132,405 and 4,946,778. Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not coil or form secondary structures. Such methods have been described in the art. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405 and 4,946,778. Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility. Anti-spike scFv antibodies have been reported (Sui et al. (2004) PNAS USA 101:2536-2541).

“Mini-antibodies” or “minibodies” will also find use with the present invention. Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region. Pack et al., (1992) Biochem 31:1579-1584. The oligomerization domain comprises self-associating α-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds. The oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein. Generally, minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al., (1992) Biochem 31:1579-1584; Cumber et al. (1992) J. Immunology 149B: 120-126.

Non-conventional means can also be used to generate and identify the antibodies of the invention. For example, a phage display library can be screened for antibodies which bind to the SARS antigens of the invention. See generally, Siegel, “Recombinant Monoclonal Antibody Technology”, Transfus. Clin. Biol. (2002) 9(1): 15-22; Sidhu, “Phage Display in Pharmaceutical Biotechnology”, Curr. Opin. Biotechnol. (2000) 11(6):610-616; Sharon, et al., “Recombinant Polyclonal Antibody Libraries”, Comb. Chem. High Throughput Screen (2000) 3(3): 185-196; and Schmitz et al., “Phage Display: A Molecular Tool for the Generation of Antibodies-Review”, Placenta, (2000) 21 SupplA: S106-12.

The antibodies of the invention may also be generated by administering the polynucleotide sequence encoding for the SARS antigen into an animal. The SARS antigen is then expressed in vivo, and antibodies specific to the SARS antigen are generated in vivo. Methods for polynucleotide delivery of the SARS antigens of the invention are discussed in section 4 below.

The antibodies of the invention are preferably specific to the SARS virus.

H. Combinations of One or More of any of the Above Approaches in a Vaccine

The compositions of the invention further comprise combinations of one or more of the compositions discussed above. For instance, the invention comprises a composition comprising an attenuated SARS virus and a subunit SARS viral antigen.

I. Combinations of SARS Antigens and Other Respiratory Virus Antigens

The invention further relates to vaccine formulations comprising one or more SARS virus antigens and one or more other respiratory virus antigens. Additional respiratory virus antigens suitable for use in the invention include antigens from influenza virus, human rhinovirus (HRV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), adenovirus, metapneumovirus, and rhinovirus. The additional respiratory virus antigen could also be from a coronavirus other than the SARS coronavirus, such as the NL63 human coronavirus (van der Hoek et al. (2004) Nature Medicine 10:368-373). Preferably, the additional respiratory virus antigen is an influenza viral antigen.

The invention may also comprise one or more bacterial or viral antigens in combination with the SARS viral antigen. Antigens may be used alone or in any combination. (See, e.g., WO 02/00249 describing the use of combinations of bacterial antigens). The combinations may include multiple antigens from the same pathogen, multiple antigens from different pathogens or multiple antigens from the same and from different pathogens. Thus, bacterial, viral, and/or other antigens may be included in the same composition or may be administered to the same subject separately. It is generally preferred that combinations of antigens be used to raise an immune response be used in combinations.

Non-limiting examples of bacterial pathogens which may be used in the invention include diphtheria (See, e.g., Chapter 3 of Vaccines, 1998, eds. Plotkin & Mortimer (ISBN 0-7216-1946-0), staphylococcus (e.g., Staphylococcus aureus as described in Kuroda et al. (2001) Lancet 357:1225-1240), cholera, tuberculosis, C. tetani, also known as tetanus (See, e.g., Chapter 4 of Vaccines, 1998, eds. Plotkin & Mortimer (ISBN 0-7216-1946-0), Group A and Group B streptococcus (including Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus pyogenes as described, for example, in Watson et al. (2000) Pediatr. Infect. Dis. J. 19:331-332; Rubin et al. (2000) Pediatr Clin. North Am. 47:269-284; Jedrzejas et al. (2001) Microbiol Mol Biol Rev 65:187-207; Schuchat (1999) Lancet 353:51-56; GB patent applications 0026333.5; 0028727.6; 015640.7; Dale et al. (1999) Infect Dis Clin North Am 13:227-1243; Ferretti et al. (2001) PNAS USA 98:4658-4663), pertussis (See, e.g., Gusttafsson et al. (1996) N. Engl. J. Med. 334:349-355; Rappuoli et al. (1991) TIBTECH 9:232-238), meningitis, Moraxella catarrhalis (See, e.g., McMichael (2000) Vaccine 19 Suppl. 1:S101-107) and other pathogenic states, including, without limitation, Neisseria meningitides (A, B, C, Y), Neisseria gonorrhoeae (See, e.g., WO 99/24578; WO 99/36544; and WO 99/57280), Helicobacter pylori (e.g., CagA, VacA, NAP, HopX, HopY and/or urease as described, for example, WO 93/18150; WO 99/53310; WO 98/04702) and Haemophilus influenza. Hemophilus influenza type B (HIB) (See, e.g., Costantino et al. (1999) Vaccine 17:1251-1263), Porphyromonas gingivalis (Ross et al. (2001) Vaccine 19:4135-4132) and combinations thereof.

Non-limiting examples of viral pathogens which may be used in the invention include meningitis, rhinovirus, influenza (Kawaoka et al., Virology (1990) 179:759-767; Webster et al., “Antigenic variation among type A influenza viruses,” p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses. Springer-Verlag, New York), respiratory syncytial virus (RSV), parainfluenza virus (PIV), rotavirus (e.g., VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 or NSP5 and other rotavirus antigens, for example as described in WO 00/26380) and the like. Antigens derived from other viruses will also find use in the present invention, such as without limitation, proteins from members of the families Picomaviridae (e.g., polioviruses, etc. as described, for example, in Sutter et al. (2000) Pediatr Clin North Am 47:287-308; Zimmerman & Spann (1999) Am Fam Physician 59:113-118; 125-126); Caliciviridae; Togaviridae (e.g., rubella virus, etc.); Flaviviridae, including the genera flavivirus (e.g., yellow fever virus, Japanese encephalitis virus, serotypes of Dengue virus, tick borne encephalitis virus, West Nile virus, St. Louis encephalitis virus); pestivirus (e.g., classical porcine fever virus, bovine viral diarrhea virus, border disease virus); and hepacivirus (e.g., hepatitis A, B and C as described, for example, in U.S. Pat. Nos. 4,702,909; 5,011,915; 5,698,390; 6,027,729; and 6,297,048); Parvovirus (e.g., parvovirus B19); Coronaviridae; Reoviridae; Bimaviridae; Rhabodoviridae (e.g., rabies virus, etc. as described for example in Dressen et al. (1997) Vaccine 15 Suppl:s2-6; MMWR Morb Mortal Wkly Rep. Jan. 16, 1998; 47(1):12, 19); Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus, respiratory syncytial virus, etc. as described in Chapters 9 to 11 of Vaccines, 1998, eds. Plotkin & Mortimer (ISBN 0-7216-1946-0); Orthomyxoviridae (e.g., influenza virus types A, B and C, etc. as described in Chapter 19 of Vaccines, 1998, eds. Plotkin & Mortimer (ISBN 0-7216-1946-0),); Bunyaviridae; Arenaviridae; Retroviradae (e.g., HTLV-1; HTLV-11; HIV-1 (also known as HTLV-III, LAV, ARV, HTI,R, etc.)), including but not limited to antigens from the isolates HIVI11b, HIVSF2, HIVLAV, HIVI-AL, I-IIVMN, SF162); HIV-I CM235, HIV-I US4; HIV-2; simian immunodeficiency virus (SIV) among others. Additionally, antigens may also be derived from human papilloma virus (HPV) and the tick-borne encephalitis viruses. See, e.g. Virology, 3^(rd) Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds, 1991), for a description of these and other viruses.

Proteins may also be derived from the herpesvirus family, including proteins derived from herpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens derived from varicella zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB and gH (See, U.S. Pat. No. 4,689,225 and PCT Publication WO 89/07143); and antigens derived from other human herpesviruses such as HHV6 and HHV7. (See, e.g. Chee et al., Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp. 125-169, for a review of the protein coding content of cytomegalovirus; McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a discussion of the various HSV-1 encoded proteins; U.S. Pat. No. 5,171,568 for a discussion of HSV-1 and HSV-2 gB and gD proteins and the genes encoding therefor; Baer et al., Nature (1984) 310:207-211, for the identification of protein coding sequences in an EBV genome; and Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816, for a review of VZV). Herpes simplex virus (HSV) rgD2 is a recombinant protein produced in genetically engineered Chinese hamster ovary cells. This protein has the normal anchor region truncated, resulting in a glycosylated protein secreted into tissue culture medium. The gD2 can be purified in the CHO medium to greater than 90% purity. Human immunodeficiency virus (HIV) env-2-3 is a recombinant form of the HIV enveloped protein produced in genetically engineered Saccharomyces cerevisae. This protein represents the entire protein region of HIV gp120 but is non-glycosylated and denatured as purified from the yeast. HIV gp120 is a fully glycosylated, secreted form of gp120 produced in CHO cells in a fashion similar to the gD2 above. Additional HSV antigens suitable for use in immunogenic compositions are described in PCT Publications W0 85/04587 and W0 88/02634, the disclosures of which are incorporated herein by reference in their entirety. Mixtures of gB and gD antigens, which are truncated surface antigens lacking the anchor regions, are particularly preferred.

Antigens from the hepatitis family of viruses, including hepatitis A virus (HAV) (See, e.g., Bell et al. (2000) Pediatr Infect Dis. J. 19:1187-1188; Iwarson (1995) APMIS 103:321-326), hepatitis B virus (HBV) (See, e.g., Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80), hepatitis C virus (HCV) (See, e.g., PCT/US88/04125, published European application number 318216), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), can also be conveniently used in the techniques described herein. By way of example, the viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. Also included in the invention are molecular variants of such polypeptides, for example as described in PCT/US99/31245; PCT/US99/31273 and PCT/US99/31272. The HCV genome encodes several viral proteins, including E1 (also known as E) and E2 (also known as E2/NSI) and an N-terminal nucleocapsid protein (termed “core”) (see, Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV proteins, including E1 and E2). Similarly, the sequence for the δ-antigen from HDV is known (see, e.g., U.S. Pat. No. 5,378,814) and this antigen can also be conveniently used in the present composition and methods. Additionally, antigens derived from HBV, such as the core antigen, the surface antigen, SAg, as well as the presurface sequences, pre-S1 and pre-S2 (formerly called pre-S), as well as combinations of the above, such as SAg/pre-S1, SAg/pre-S2, SAg/pre-S1/pre-S2, and pre-S1/pre-S2, will find use herein. See, e.g., “HBV Vaccines—from the laboratory to license: a case study” in Mackett, M. and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176, for a discussion of HBV structure; and U.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513, incorporated herein by reference in their entireties; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum et al., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991) 65:5457-5464. Each of these proteins, as well as antigenic fragments thereof, will find use in the present composition and methods.

Influenza virus is another example of a virus for which the present invention will be particularly useful. Specifically, the envelope glycoproteins HA and NA of influenza A are of particular interest for generating an immune response. Numerous HA subtypes of influenza A have been identified (Kawaoka et al., Virology (1990) 179:759-767; Webster et al., “Antigenic variation among type A influenza viruses,” p. 127-168. In: P. Palese and D. W. Kingsbury (ed.), Genetics of influenza viruses. Springer-Verlag, New York). Thus, proteins derived from any of these isolates can also be used in the compositions and methods described herein.

Non-limiting examples of parasitic antigens include those derived from organisms causing malaria and Lyme disease.

The methods of the invention comprise administering an immunogenic composition comprising a SARS viral antigen (including one or more of an inactivated SARS virus, an attenuated SARS virus, a split SARS virus preparation or a recombinant or purified subunit formulation of one or more SARS viral antigens) to an animal. The immunogenic compositions used in the invention can comprise an immunologically effective amount of the SARS viral antigen. An “immunologically effective amount” is an amount sufficient to allow the mammal to raise an immune response to the SARS antigen.

The immune response preferably involves the production of antibodies specific to the SARS antigen. The amount of antibodies produced will vary depending on several factors including the animal used, the presence of an adjuvant, etc.

The immunogenic compositions of the invention may further comprise one or more adjuvants.

The immunogenic compositions of the invention may be administered mucosally. Suitable routes of mucosal administration include oral, intranasal, intragastric, pulmonary, intestinal, rectal, ocular and vaginal routes. The immunogenic composition may be adapted for mucosal administration. For instance, where the composition is for oral administration, it may be in the form of tablets or capsules, optionally enteric-coated, liquid, transgenic plants, etc. Where the composition is for intranasal administration, it may be in the form of a nasal spray, nasal drops, gel or powder.

The immunogenic compositions of the invention may be administered parenterally. Suitable routes of parenteral administration include intramuscular (IM), subcutaneous, intravenous, intraperitoneal, intradermal, transcutaneous, and transdermal (see e.g., International patent application WO 98/20734) routes, as well as delivery to the interstitial space of a tissue. The immunogenic composition may be adapted for parenteral administration, for instance in the form of an injectable that may be sterile and pyrogen free.

Vaccines of the invention may be administered in conjunction with other immunoregulatory agents. In particular, compositions will usually include an adjuvant. Preferred further adjuvants include, but are not limited to, one or more of the following set forth below:

A. Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts. The invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphoshpates, orthophosphates), sulphates, etc. (e.g. see chapters 8 & 9 of Vaccine design: the subunit and adjuvant approach (1995) Powell & Newman. ISBN 0-306-44867-X.), or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt. See WO00/23105.

B. Oil-Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). See WO90/14837. See also, Frey et al., “Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non-elderly adults”, Vaccine (2003) 21:4234-4237.

Particularly preferred adjuvants for use in the compositions are submicron oil-inwater emulsions. Preferred submicron oil-in-water emulsions for use herein are squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80™ (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span 85™ (sorbitan trioleate), and, optionally, N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphophoryloxy)-ethylamine (MTP-PE), for example, the submicron oil-in-water emulsion known as “MF59” (International Publication No. WO 90/14837; U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entireties; and Ott et al., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/v Squalene (e.g., 4.3%), 0.25-0.5% w/v Tween 80™, and 0.5% w/v Span 85™ and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PE may be present in an amount of about 0-500 μg/dose, more preferably 0-250 μg/dose and most preferably, 0-100 μg/dose. As used herein, the term “MF59-0” refers to the above submicron oil-in-water emulsion lacking MTP-PE, while the term MF59-MTP denotes a formulation that contains MTP-PE. For instance, “MF59-100” contains 100 μg MTP-PE per dose, and so on. MF69, another submicron oil-in-water emulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v Tween 80™, and 0.75% w/v Span 85™ and optionally MTP-PE. Yet another submicron oil-in-water emulsion is MF75, also known as SAF, containing 10% squalene, 0.4% Tween 80™, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidized into a submicron emulsion. MF75-MTP denotes an MF75 formulation that includes MTP, such as from 100-400 μg MTP-PE per dose.

Submicron oil-in-water emulsions, methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO 90114837 and U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporated herein by reference in their entireties.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants in the invention.

C. Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.

Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-LC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. A method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulations may also comprise a sterol, such as cholesterol (see WO 96/33739).

Combinations of saponins and cholesterols can be used to form unique particles called Immunostimulating Complexs (ISCOMs). ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of Quil A, QHA and QHC. ISCOMs are further described in EP 0 109 942, WO 96/11711 and WO 96/33739. Optionally, the ISCOMS may be devoid of additional detergent. See WO00/07621.

A review of the development of saponin based adjuvants can be found at Barr, et al., “ISCOMs and other saponin based adjuvants”, Advanced Drug Delivery Reviews (1998) 32:247-271. See also Sjolander, et al., “Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines”, Advanced Drug Delivery Reviews (1998) 32:321-338.

D. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as:

(1) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such “small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529. See Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.

(2) Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in Meraldi et al., “OM-174, a New Adjuvant with a Potential for Human Use, Induces a Protective Response with Administered with the Synthetic C-Terminal Fragment 242-310 from the circumsporozoite protein of Plasmodium berghei”, Vaccine (2003) 21:2485-2491; and Pajak, et al., “The Adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo”, Vaccine (2003) 21:836-842.

(3) Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded. Optionally, the guanosine may be replaced with an analog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al., “Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles”, Nucleic Acids Research (2003) 31(9): 2393-2400; WO 02/26757 and WO 99/62923 for examples of possible analog substitutions. The adjuvant effect of CpG oligonucleotides is further discussed in Krieg, “CpG motifs: the active ingredient in bacterial extracts?”, Nature Medicine (2003) 9(7): 831-835; McCluskie, et al., “Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA”, FEMS Immunology and Medical Microbiology (2002) 32:179-185; WO 98/40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S. Pat. No. 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. See Kandimalla, et al., “Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpG DNAs”, Biochemical Society Transactions (2003) 31 (part 3): 654-658. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, et al., “CpG-A-Induced Monocyte IFN-gamma-Inducible Protein-10 Production is Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha”, J. Immunol. (2003) 170(8):4061-4068; Krieg, “From A to Z on CpG”, TRENDS in Immunology (2002) 23(2): 64-65 and WO 01/95935. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3′ ends to form “immunomers”. See, for example, Kandimalla, et al., “Secondary structures in CpG oligonucleotides affect immunostimulatory activity”, BBRC (2003) 306:948-953; Kandimalla, et al., “Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic GpG DNAs”, Biochemical Society Transactions (2003) 31(part 3):664-658; Bhagat et al., “CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents” BBRC (2003) 300:853-861 and WO 03/035836.

(4) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (i.e., E. coli heat labile enterotoxin “LT), cholera (“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO 95/17211 and as parenteral adjuvants in WO 98/42375. Preferably, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use of ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT-R72, as adjuvants can be found in the following references, each of which is specifically incorporated by reference herein in their entirety: Beignon, et al., “The LTR72 Mutant of Heat-Labile Enterotoxin of Escherichia coli Enahnces the Ability of Peptide Antigens to Elicit CD4+ T Cells and Secrete Gamma Interferon after Coapplication onto Bare Skin”, Infection and Immunity (2002) 70(6):3012-3019; Pizza, et al., “Mucosal vaccines: non-toxic derivatives of LT and CT as mucosal adjuvants”, Vaccine (2001) 19:2534-2541; Pizza, et al., “LTK63 and LTR72, two mucosal adjuvants ready for clinical trials” Int. J. Med. Microbiol (2000) 290(4-5):455-461; Scharton-Kersten et al., “Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits and Unrelated Adjuvants”, Infection and Immunity (2000) 68(9):5306-5313; Ryan et al., “Mutants of Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Th1 and Th2 Cells” Infection and Immunity (1999) 67(12):6270-6280; Partidos et al., “Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides”, Immunol. Lett. (1999) 67(3):209-216; Peppoloni et al., “Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines”, Vaccines (2003) 2(2):285-293; and Pine et al., (2002) “Intranasal immunization with influenza vaccine and a detoxified mutant of heat labile enterotoxin from Escherichia coli (LTK63)” J. Control Release (2002) 85(1-3):263-270. Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in Domenighini et al., Mol. Microbiol (1995) 15(6): 1165-1167, specifically incorporated herein by reference in its entirety.

E. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in the invention. Suitable bioadhesives include esterified hyaluronic acid microspheres (Singh et al. (2001) J. Cont. Rele. 70:267-276) or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention. E.g., WO99/27960.

G. Microparticles

Microparticles may also be used as adjuvants in the invention. Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes

Examples of liposome formulations suitable for use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0 626 169.

I. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters. WO99/52549. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/21152).

Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

J. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andrianov et al., “Preparation of hydrogel microspheres by coacervation of aqueous polyphophazene solutions”, Biomaterials (1998) 19(1-3):109-115 and Payne et al., “Protein Release from Polyphosphazene Matrices”, Adv. Drug. Delivery Review (1998) 31(3):185-196.

K. Muramyl peptides

Examples of muramyl peptides suitable for use as adjuvants in the invention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylaamine MTP-PE).

L. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues, described further in Stanley, “Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential” Clin Exp Dermatol (2002) 27(7):571-577 and Jones, “Resiquimod 3M”, Curr Opin Investig Drugs (2003) 4(2):214-218

M. Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvants in the invention. These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome. The viral proteins may be recombinantly produced or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein p1). VLPs are discussed further in WO 03/024480, WO 03/024481, and Niukura et al., “Chimeric Recombinant Hepatitis E Virus-Like Particles as an Oral Vaccine Vehicle Presenting Foreign Epitopes”, Virology (2002) 293:273-280; Lenz et al., “Papillomarivurs-Like Particles Induce Acute Activation of Dendritic Cells”, Journal of Immunology (2001) 5246-5355; Pinto, et al., “Cellular Immune Responses to Human Papillomavirus (HPV)-16 L1 Healthy Volunteers Immunized with Recombinant HPV-16 L1 Virus-Like Particles”, Journal of Infectious Diseases (2003) 188:327-338; and Gerber et al., “Human Papillomavrisu Virus-Like Particles Are Efficient Oral immunogens when Coadministered with Escherichia coli Heat-Labile Entertoxin Mutant R192G or CpG”, Journal of Virology (2001) 75(10):4752-4760. Virosomes are discussed further in, for example, Gluck et al., “New Technology Platforms in the Development of Vaccines for the Future”, Vaccine (2002) 20:B10-B16.

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions may be used in the invention:

(1) a saponin and an oil-in-water emulsion (WO99/11241);

(2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3dMPL) (see WO 94/00153);

(3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3dMPL)+a cholesterol;

(4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally +a sterol) (WO98/57659);

(5) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (See European patent applications 0835318, 0735898 and 0761231);

(6) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion.

(7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); and

(8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dPML).

Aluminium salts and MF59 are preferred adjuvants for parenteral immunisation. Mutant bacterial toxins are preferred mucosal adjuvants.

As mentioned above, adjuvants suitable for use in the invention may also include one or more of the following:

E. coli heat-labile enterotoxin (“LT”), or detoxified mutants thereof, such as the K63 or R72 mutants;

cholera toxin (“CT”), or detoxified mutants thereof;

microparticles (i.e., a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to ˜30 μm in diameter, and most preferably 500 nm to ˜10 μm in diameter) formed from materials that are biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone etc.);

a polyoxyethylene ether or a polyoxyethylene ester (see International patent application WO 99/52549);

a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (see International patent application WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (see International patent application WO 01/21152);

chitosan (e.g. International patent application WO 99/27960)

an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin (see International patent application WO 00/62800)

immunostimulatory double stranded RNA.

aluminum compounds (e.g. aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, oxyhydroxide, orthophosphate, sulfate etc. (e.g. see chapters 8 & 9 of Vaccine design: the subunit and adjuvant aproach, eds. Powell & Newman, Plenum Press 1995 (ISBN 0-306-44867-X) (hereinafter “Vaccine design”), or mixtures of different aluminum compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous etc.), and with adsorption being preferred;

MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer) (see Chapter 10 of Vaccine design; see also International patent application WO 90/14837);

liposomes (see Chapters 13 and 14 of Vaccine design);

ISCOMs (see Chapter 23 of Vaccine design);

SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion (see Chapter 12 of Vaccine design);

Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™);

saponin adjuvants, such as QuilA or QS21 (see Chapter 22 of Vaccine design), also known as Stimulon™;

ISCOMs, which may be devoid of additional detergent (WO 00/07621);

complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA);

cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, tumor necrosis factor, etc. (see Chapters 27 & 28 of Vaccine design);

monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (e.g. chapter 21 of Vaccine design);

combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (European patent applications 0835318, 0735898 and 0761231);

oligonucleotides comprising CpG motifs (see Krieg (2000) Vaccine, 19:618-622; Krieg (2001) Curr. Opin. Mol. Ther., 2001, 3:15-24; WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581, etc.) i.e. containing at least one CG dinucleotide,

a polyoxyethylene ether or a polyoxyethylene ester (International patent application WO99/52549);

a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (International patent application WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152);

an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin (WO00/62800);

an immunostimulant and a particle of metal salt (International patent application WO00/23105);

a saponin and an oil-in-water emulsion (WO 99/11241);

a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) (WO 98/57659).

Other adjuvants suitable for mucosal or parenteral administration are also available (e.g. see chapter 7 of Vaccine design: the subunit and adjuvant aproach, eds. Powell & Newman, Plenum Press 1995 (ISBN 0-306-44867-X).

Mutants of LT are preferred mucosal adjuvants, in particular the “K63” and “R72” mutants (e.g. see International patent application WO 98/18928), as these result in an enhanced immune response.

Microparticles are also preferred mucosal adjuvants. These are preferably derived from a poly(a-hydroxy acid), in particular, from a poly(lactide) (“PLA”), a copolymer of D,L-lactide and glycolide or glycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or “PLGA”), or a copolymer of D,L-lactide and caprolactone. The microparticles may be derived from any of various polymeric starting materials which have a variety of molecular weights and, in the case of the copolymers such as PLG, a variety of lactide:glycolide ratios, the selection of which will be largely a matter of choice, depending in part on the coadministered antigen.

The SARS virus (inactivated or attenuated), viral antigens, antibodies or adjuvants of the invention may be entrapped within the microparticles, or may be adsorbed to them. Entrapment within PLG microparticles is preferred. PLG microparticles are discussed in further detail in Morris et al., (1994), Vaccine, 12:5-11, in chapter 13 of Mucosal Vaccines, eds. Kiyono et al., Academic Press 1996 (ISBN 012410587), and in chapters 16 & 18 of Vaccine design: the subunit and adjuvant aproach, eds. Powell & Newman, Plenum Press 1995 (ISBN 0-306-44867-X).

LT mutants may advantageously be used in combination with microparticle-entrapped antigen, resulting in significantly enhanced immune responses.

Aluminium compounds and MF59 are preferred adjuvants for parenteral use.

The composition may include an antibiotic.

The immunogenic compositions of the invention may be administered in a single dose, or as part of an administration regime. The regime may include priming and boosting doses, which may be administered mucosally, parenterally, or various combinations thereof.

The methods of the invention further comprise treating or preventing a SARS virus-related disease by administering to an animal a composition comprising an effective amount of the antibodies of the invention. An “effective amount” of the antibodies of the invention is an amount sufficient to provide passive immunization protection or treatment to the animal. Preferably, the antibodies of the invention are specific to the SARS viral antigen.

Methods of treatment may combine both immunogenic compositions and antibody compositions. Accordingly the invention comprises a method for treating or preventing a SARS virus-related disease comprising administering an immunogenic composition comprising an immunologically effective amount of a SARS viral antigen and administering an effective amount of antibodies specific to SARS viral antigen. The immunogenic composition and the antibodies may be administered together or separately. The invention further comprises a composition comprising an immunogenic composition comprising an immunologically effective amount of a SARS viral antigen and further comprising an effective amount of antibodies specific to a SARS viral antigen.

The SARS viral antigens and antibodies of the invention may also be administered in polynucleotide form. The SARS viral antigens and/or antibody proteins are then expressed in vivo.

The SARS viral antigens and the antibodies of the invention can also be delivered using one or more gene vectors, administered via nucleic acid immunization or the like using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. The constructs can be delivered (e.g., injected) either subcutaneously, epidermally, intradermally, intramuscularly, intravenous, mucosally (such as nasally, rectally and vaginally), intraperitoneally, orally or combinations thereof. Intramuscular injection of 25 μg plasmid DNA encoding spike antigens, in 200 μl PBS pH 7.4, at weeks 0, 3 and 6, has been described for mice by Yang et al. (2004) Nature 428:561-564.

An exemplary replication-deficient gene delivery vehicle that may be used in the practice of the present invention is any of the alphavirus vectors, described in, for example, U.S. Pat. Nos. 6,342,372; 6,329,201 and International Publication WO 01/92552.

A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller & Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie & Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Adenoviral delivery of codon-optimsed versions of the genes encoding SARS coronavirus structural antigens spike S1, membrane protein and nucleocapsid protein has been investigated in rhesus macaques and found to invoke a strong neutralizing antibody response (Gao et al. (2003) Lancet 362(9399):1895-1896).

Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering polynucleotides, mucosally and otherwise, is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference) as well as the vaccinia virus and avian poxviruses. By way of example, vaccinia virus recombinants expressing the genes can be constructed as follows. The DNA encoding the SARS antigen or antibody or antibody coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells that are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome. The resulting TK⁻ recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver genes encoding the SARS viral antigens or antibodies of the invention. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545. Picornavirus-derived vectors can also be used. (See, e.g., U.S. Pat. Nos. 5,614,413 and 6,063,384).

Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

A vaccinia based infection/transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, a SARS viral antigen or antibody expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase that in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No. 5,135,855.

The immunogenic compositions of the invention may further comprise diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like may be included in the immunogenic composition.

The immunogenic compositions used in the invention can be administered to an animal. Animals suitable for use in the methods of the invention include humans and other primates, including non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses, domestic animals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese and the like. Animals suitable for use in the invention can be of any age, including both adult and newborn. Transgenic animals can also be used in the invention.

The immunogenic compositions of the invention can be-used to treat or prevent SARS virus-related diseases.

The compositions of the invention are preferably pharmaceutically acceptable and pharmacologically acceptable. In particularly, the compositions are preferably not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

Pharmaceutically acceptable salts can also be used in compositions of the invention, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art. Compositions of the invention can also contain liquids or excipients, such as water, saline, glycerol, dextrose, ethanol, or the like, singly or in combination, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes can also be used as a carrier for a composition of the invention.

SARS specific reagents and analytical assays may be used in the manufacture and testing of the vaccines of the invention. Such analytical assays include, for example: 1) virus titration and plaque assays for quantitation of infectious virus particles, 2) a neutralization assay with constant virus and varying serum dilutions, 3) a two step RT-PCR system (Light Cycler-Roche) for detection of negative strand viral RNA, with the target sequence located within the N gene, providing highest possible sensitivity, and 4) ELISA and western blot assays for detection and qualification of viral proteins.

In addition, rabbit polyclonal antiserum has been generated to obtain antibody reagents (and demonstrate induction of neutralizing antibodies) against the SARS-CoV. A sample protocol for generating such reagents is set forth below. The virus is first cultivated in suitable cell culture, such as Vero cells, and pelleted through a 20% sucrose (w/v) cushion. The pellet is then subjected to a glycerol potassium-tartrate gradient for further purification. The virus-containing fraction is then diluted and pelleted by ultracentrifugation. The pellet is then dissolved in PBS and the virus is inactivated with C₃H₄O₂ (beta-propiolactone, BPL). Two rabbits are immunized subcutaneously (SC) on day 0, 14, and 2 8 with 1×10⁹ inactivated viral particles mixed with IFA as adjuvant. Rabbits are bled on days 0 (p,re-inoculation), 13, 28, and 35 (1 week after 3rd immunization). Sera obtained from this protocol were tested for their reactivity against SARS-CoV proteins in western blots and found to react with the major structural proteins spike (S), membrane (M), and nucleocapsid (N).

J. Emerging Coronavirus Vaccines

The SARS epidemic has lead to increased awareness of viral infections caused by coronaviruses. The vaccines of the invention may be adapted to prevent or treat emerging strains of coronavirus, including emerging strains of SARS virus.

The invention provides a vaccine comprising an inactivated (or killed) human coronavirus, an attenuated human coronavirus, a split human coronavirus preparation, or a recombinant or purified subunit formulation of one or more antigens from a human coronavirus, wherein the human coronavirus is not the SARS coronavirus. Optionally, the human coronavirus is not the 229E coronavirus. Optionally, the human coronavirus is not the OC43 coronavirus. Optionally, the human coronavirus is not the NL63 coronavirus. Thus the invention provides a vaccine as defined above, wherein the human coronavirus is not the SARS coronavirus, is not the 229E coronavirus, is not the OC43 coronavirus and is not the NL63 coronavirus. Such vaccines are useful for preventing and/or treating emerging human coronavirus infections.

The invention also provides a vaccine comprising: (a) an inactivated (or killed) human coronavirus, an attenuated human coronavirus, a split human coronavirus preparation, or a recombinant or purified subunit formulation of one or more antigens from a human coronavirus, wherein the human coronavirus is not the SARS coronavirus, as defined above; and (b) an inactivated (or killed) human coronavirus, an attenuated human coronavirus, a split human coronavirus preparation, or a recombinant or purified subunit formulation of one or more antigens from a human coronavirus, wherein the human coronavirus is the SARS coronavirus. Such vaccines are useful for preventing and/or treating both SARS and other human coronaviruses.

As well as providing vaccines comprising antigens from more than one type of coronavirus, the invention also provides vaccines comprising antigens from more than one strain of the same coronavirus e.g. different strains of the SARS coronavirus, or different strains of a coronavirus other than the SARS coronavirus. In one embodiment, the vaccine comprises antigens from at least two strains of coronavirus, or at least three strains of coronavirus. In one embodiment, the vaccine comprises antigens from at least two types of coronavirus. In one embodiment, the vaccine comprises at least one antigen from each of the known types of coronaviruses (type I, type II and type III). Such vaccines follow the model of current influenza vaccines.

The selection of coronaviruses and/or coronavirus strains for use in vaccines of the invention can be based on various criteria. For instance, selection may be based on viruses and/or strains that have been detected in the geographical region (e.g. northern or southern hemisphere, a particular country, etc.) where the vaccine targeted. Selection may be based on the results of animal surveillance e.g. of viruses detected in cat populations. Selection may be based on the results of clinical surveillance e.g. of viruses detected in patients hospitalized with respiratory infection. Selection may be performed every year e.g. prior to winter. Vaccines may also be administered yearly, again following the model of current influenza vaccines.

Preferred vaccines are sufficiently immunogenic to provide a neutralizing immune response, and more preferably a protective and/or therapeutic immune response. Particularly preferred vaccines meet the efficacy requirements that may be specified by the WHO from time to time.

A preferred subunit antigen for inclusion in vaccines of the invention is a purified spike protein, more preferably in oligomeric (e.g. trimeric) form. The spike protein may or my not be cleaved e.g. into its S1 and S2 products.

The techniques disclosed above for selecting viruses and/or strains for production of vaccines can also be used to select appropriate viruses and/or strains from which HR1 and HR2 sequences can be obtained for providing therapeutic peptides, as disclosed above.

III. Diagnostic Compositions and Methods of the Invention

The invention provides methods for detecting the SARS coronavirus. Detection in patient samples can be used to detect and diagnose infections by the virus. Detection in donated blood can be used to prevent inadvertent transmission of the virus during blood transplant procedures Detection methods fall into three main categories: detection of SARS virus nucleic acids; detection of SARS virus proteins; and detection of anti-SARS virus immune responses. The invention provides all such methods.

As used herein when referring to nucleotide sequences, particularly oligonucleotide probes and primers, “similar” sequences includes those sequences that are at least 90% identical to known SARSV genomic sequence and includes sequences that are at least 95% identical, at least 99% identical and 100% identical to the SARSV genomic sequence over the length of the probe or primer.

As used herein, the term “target nucleic acid region” or “target nucleic acid” denotes a nucleic acid molecule with a “target sequence” to be amplified. The target nucleic acid may be either single-stranded or double-stranded and may include other sequences besides the target sequence, which may not be amplified. The term “target sequence” refers to the particular nucleotide sequence of the target nucleic acid which is to be amplified. The target sequence may include a probe-hybridizing region contained within the target molecule with which a probe will form a stable hybrid under desired conditions. The “target sequence” may also include the complexing sequences to which the oligonucleotide primers complex and be extended using the target sequence as a template. Where the target nucleic acid is originally single-stranded, the term “target sequence” also refers to the sequence complementary to the “target sequence” as present in the target nucleic acid. If the “target nucleic acid” is originally double-stranded, the term “target sequence” refers to both the plus (+) and minus (−) strands.

The term “primer” or “oligonucleotide primer” as used herein, refers to an oligonucleotide which acts to initiate synthesis of a complementary DNA strand when placed under conditions in which synthesis of a primer extension product is induced i.e. in the presence of nucleotides and a polymerization-inducing agent such as a DNA or RNA polymerase and at suitable temperature, pH, metal concentration, and salt concentration. The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. This denaturation step is typically effected by heat, but may alternatively be carried out using alkali, followed by neutralization. Thus, a “primer” is complementary to a template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the addition of covalently bonded bases linked at its 3′ end complementary to the template in the process of DNA synthesis.

As used herein, the term “probe” or “oligonucleotide probe” refers to a structure comprised of a polynucleotide, as defined above, that contains a nucleic acid sequence complementary to a nucleic acid sequence present in the target nucleic acid analyte. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. When an “oligonucleotide probe” is to be used in a 5′ nuclease assay, such as the TaqMan™ technique, the probe will contain at least one fluorescer and at least one quencher which is digested by the 5′ endonuclease activity of a polymerase used in the reaction in order to detect any amplified target oligonucleotide sequences. In this context, the oligonucleotide probe will have a sufficient number of phosphodiester linkages adjacent to its 5′ end so that the 5′ to 3′ nuclease activity employed can efficiently degrade the bound probe to separate the fluorescers and quenchers. When an oligonucleotide probe is used in the TMA technique, it will be suitably labeled, as described below.

It will be appreciated that the hybridizing sequences need not have perfect complementarity to provide stable hybrids. In many situations, stable hybrids will form where fewer than about 10% of the bases are mismatches, ignoring loops of four or more nucleotides. Accordingly, as used herein the term “complementary” refers to an oligonucleotide that forms a stable duplex with its “complement” under assay conditions, generally where there is about 90% or greater homology.

The terms “hybridize” and “hybridization” refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing. Where a primer “hybridizes” with target (template), such complexes (or hybrids) are sufficiently stable to serve the priming function required by e.g. the DNA polymerase to initiate DNA synthesis.

Stringent hybridization conditions will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are typically greater than 22° C., more typically greater than about 30° C., and preferably in excess of about 37° C. Longer fragments may require higher hybridization temperatures for specific hybridization. Other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, and the combination of parameters used is more important than the absolute measure of any one alone. Other hybridization conditions which may be controlled include buffer type and concentration, solution pH, presence and concentration of blocking reagents to decrease background binding such as repeat sequences or blocking protein solutions, detergent type(s) and concentrations, molecules such as polymers which increase the relative concentration of the polynucleotides, metal ion(s) and their concentration(s), chelator(s) and their concentrations, and other conditions known in the art. Less stringent, and/or more physiological, hybridization conditions are used where a labeled polynucleotide amplification product cycles on and off a substrate linked to a complementary probe polynucleotide during a real-time assay which is monitored during PCR amplification such as a molecular beacon assay. Such less stringent hybridization conditions can also comprise solution conditions effective for other aspects of the method, for example reverse transcription or PCR.

As used herein, a “biological sample” refers to a sample of tissue, cells or fluid isolated from a subject, that commonly includes antibodies produced by the subject. Typical samples include but are not limited to, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, mucous, milk, blood cells, organs, tissues, biopsies (e.g. lung, liver, kidney) and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium e.g. recombinant cells, and cell components. Other samples that may be used for diagnosis include stool samples and nasopharyngeal aspirates.

The term “antibody” encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)₂ and F(ab) fragments; Fv molecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); oligobodies; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Packet al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and UK Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.

As used herein, the term “monoclonal antibody” refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins.

Methods of making polyclonal and monoclonal antibodies are known in the art. Polyclonal antibodies are generated by immunizing a suitable animal, such as a mouse, rat, rabbit, sheep or goat, with an antigen of interest. In order to enhance immunogenicity, the antigen can be linked to a carrier prior to immunization. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Furthermore, the antigen may be conjugated to a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc., in order to enhance the immunogenicity thereof.

Rabbits, sheep and goats are preferred for the preparation of polyclonal sera when large volumes of sera are desired. These animals are good design choices also because of the availability of labeled anti-rabbit, anti-sheep and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant (“FCA”), and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). The animal is generally boosted 2-6 weeks later with one or more injections of the antigen in saline, preferably using Freund's incomplete adjuvant (“FIA”). Antibodies may also be generated by in vitro immunization, using methods known in the art. Polyclonal antisera is then obtained from the immunized animal.

Monoclonal antibodies are generally prepared using the method of Kohler & Milstein (1975) Nature 256:495-497, or a modification thereof, as described above.

Nucleic Acid Detection Methods

There are many well known methods of amplifying targeted sequences, such as the polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), the ligase chain reaction (LCR), the strand displacement amplification (SDA), and the nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA) to name a few. These methods are described generally in the following references: (PCR) U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; (RT-PCR) U.S. Pat. Nos. 5,310,652, 5,322,770; (LCR) EP Application No., 320,308 published Jun. 14, 1989; (SDA) U.S. Pat. Nos. 5,270,184, and 5,455,166 and “Empirical Aspects of Strand Displacement Amplification” by G. T. Walker in PCR Methods and Applications, 3(1):1-6 (1993), Cold Spring Harbor Laboratory Press; (TMA) U.S. Pat. No. 5,399,491, and (NASBA) “Nucleic Acid Sequence-Based Amplification (NASBA™)” by L. Malek et al., Ch. 36 in Methods in Molecular Biology, Vol. 28: Protocols for Nucleic Acid Analysis by Nonradioactive Probes, 1994 Ed. P. G. Isaac, Humana Press, Inc., Totowa, N.J. PCR methods may include variations that permit quantitation of the target sequence, for example, by real time PCR analysis (e.g., as described in U.S. Pat. Nos. 5,210,015, 5,487,972, 5,994,056, 6,171,785 inter alia). (Each of the above references are hereby incorporated by reference).

One embodiment of the method of the invention for detecting the presence of SARS virus in a sample comprises providing a sample suspected of containing a SARS virus nucleic acid target, amplifying a template sequence contained within said SARS virus nucleic acid target by any known technique of nucleic acid amplification, including any of those mentioned herein, using the oligonucleotide primers described herein, particularly those primers comprising the kits described herein, and detecting the amplified template sequence, wherein the presence of the amplified template sequence indicates the presence of SARS virus in said sample.

Amplification techniques generally involve the use of two primers. Where a target sequence is single-stranded, the techniques generally involve a preliminary step in which a complementary strand is made in order to give a double-stranded target. The two primers hybridize to different strands of the double-stranded target and are then extended. The extended products can serve as targets for further rounds of hybridization/extension. The net effect is to amplify a template sequence within the target, the 5′ and 3′ termini of the template being defined by the locations of the two primers in the target. As an alternative, if one or both of the primers contains a promoter sequence then the target can be amplified (by transcription) using a RNA polymerase (as in TMA).

The present invention provides methods and kits for amplifying and/or detecting a template or target sequence in the SARSV viral nucleic acid. The invention provides a kit comprising primers for amplifying a template sequence contained within a SARSV nucleic acid target, the kit comprising a first primer and a second primer, wherein the first primer comprises a sequence substantially complementary to a portion of said template sequence and the second primer comprises a sequence substantially complementary to a portion of the complement of said template sequence, wherein the sequences within said primers which have substantial complementarity define the termini of the template sequence to be amplified.

Kits of the invention may further comprise a probe which is substantially complementary to the template sequence and/or to its complement and which can hybridize thereto. This probe can be used in a hybridization technique to detect amplified template, or to isolate (i.e. “capture) the amplified template or the original target nucleic acid.

Kits of the invention may further comprise primers and/or probes for generating and detecting an internal standard, in order to aid quantitative measurements (e.g Fille et al. 1997 Biotechniques 23:34-36).

Kits of the invention may further comprise a DNA polymerase, which will generally be a thermostable DNA polymerase where a non-isothermal amplification process is to be used. The kits may also comprise supplies of dNTPs, a magnesium salt (e.g. MgCl₂), buffer solutions, etc.

Kits of the invention may comprise more than one pair of primers (e.g. for nested amplification), and one primer may be common to more than one primer pair. The kit may also comprise more than one probe.

Oligomer Probes and Primers

In connection with the nucleic acid detection methods of the present invention described above, oligomers having sequence similarity, or complementarity, to the SARSV genome are useful. The SARSV genome sequences mentioned herein may be used to produce probes and primers which can be used in assays for the detection of nucleic acids in test samples. The probes may be designed from conserved nucleotide regions of the polynucleotides of interest or from non-conserved nucleotide regions of the polynucleotide of interest. The design of such probes for optimization in assays is within the skill of those of ordinary skill in the art. Generally, nucleic acid probes are developed from non-conserved or unique regions when maximum specificity is desired, and nucleic acid probes are developed from conserved regions when assaying for nucleotide regions that are closely related to, for example, different members of a multi-gene family or in related species like mouse and man.

Using as a basis the SARSV genome which can be found as described herein, and/or preferably conserved regions of the SARSV genome, and/or the particularly described primer and probe sequences as disclosed herein, oligomers of approximately 8 nucleotides or more can be prepared which hybridize with the positive strand(s) of SARSV RNA or its complement, as well as to SARSV cDNAs. These oligomers can serve as probes for the detection (including isolation and/or labeling) of polynucleotides which contain SARSV nucleotide sequences, and/or as primers for the transcription and/or replication of targeted SARSV sequences. The oligomers contain a targeting polynucleotide sequence, which is comprised of nucleotides which are complementary to a target SARSV nucleotide sequence; the sequence is of sufficient length and complementarity with the SARSV sequence to form a duplex which has sufficient stability for the purpose intended. For example, if the purpose is the isolation, via immobilization, of an analyte containing a target SARSV sequence, the oligomers would contain a polynucleotide region which is of sufficient length and complementarity to the targeted SARSV sequence to afford sufficient duplex stability to immobilize the analyte on a solid surface, via its binding to the oligomers, under the isolation conditions. For example, also, if the oligomers are to serve as primers for the transcription and/or replication of target SARSV sequences in an analyte polynucleotide, the oligomers would contain a polynucleotide region of sufficient length and complementarity to the targeted SARSV sequence to allow the polymerizing agent to continue replication from the primers which are in stable duplex form with the target sequence, under the polymerizing conditions. For example, also, if the oligomers are to be used as label probes, or are to bind to multimers, the targeting polynucleotide region would be of sufficient length and complementarity to form stable hybrid duplex structures with the label probes and/or multimers to allow detection of the duplex. The oligomers may contain a minimum of about 4 contiguous nucleotides which are complementary to targeted SARSV sequence; usually the oligomers will contain a minimum of about 8 contiguous nucleotides which are complementary to the targeted SARSV sequence, and preferably will contain a minimum of about 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides and up to about 50, 75, 100, 200 contiguous nucleotides or more, which are complementary to the targeted SARSV sequence.

Typically, for use in the amplification based methods (for example, PCR, RT-PCR, TMA) oligomers will be used as primer sets such that one member of the primer set has sequence similarity or complementarity to a more conserved (among coronaviruses) portion of the SARSV genome and the other member of the primer set has sequence similarity or complementarity to a less conserved portion. The primer sets can be used to amplify the target region in ways that are well known in the art. Typically, the 5′ untranslated region (5′UTR) and the 3′ untranslated region (3′UTR) are among the most conserved regions. FIG. 8 shows an alignment of the 5′UTR of several coronaviruses. FIG. 10 shows an alignment of the 3′UTR of several coronaviruses. FIGS. 9 and 11 show the sequences of preferred primers for amplification of the 5′UTR and 3′UTR, respectively. Other primers and probes can readily be designed based on the sequence alignments provided herein.

The oligomer, however, need not consist only of the sequence which is complementary to the targeted SARSV sequence. It may contain in addition, nucleotide sequences (e.g. promoters) or other moieties which are suitable for the purposes for which the oligomers are used. For example, if the oligomers are used as primers for the amplification of SARSV sequences via, for example, PCR, they may contain sequences which, when in duplex, form restriction enzyme sites which facilitate the cloning of the amplified sequences. For example, also, if the oligomers are to be used as “capture probes” in hybridization assays, they would contain in addition a binding partner which is coupled to the oligomer containing the nucleotide sequence which is complementary to the targeted SARSV sequence. Other types of moieities or sequences which are useful of which the oligomers may be comprised or coupled to, are those which are known in the art to be suitable for a variety of purposes, including the labeling of nucleotide probes.

Table 4 (SEQ ID NOS: 1021-6020) shows forward and reverse primers that are useful for nucleic acid amplification of SARSV for diagnostic and screening methods.

Preferred primers and probes for SARS nucleic acid detection for diagnostic and screening are SEQ ID NOS: 7332-7336 (forward primers), SEQ ID NOS: 7337-7341 (reverse primers) and SEQ ID NOS: 7342-7352 (probes). These primers and probes are useful for detection of sequences in the 3′ UTR.

Any of the above forward primers may be used in combination with any of the above reverse primers for amplification of SARSV nucleic acid. The amplified product may be detected (or captured) with any of the above probes. Particularly preferred combinations of forward and reverse primers and the probes for detecting the amplified product include: Forward SEQ ID NO: 7332 with reverse SEQ ID NO: 7337, 7338, 7339 or 7341 and probe SEQ ID NO: 7342; forward SEQ ID NO: 7333 or 7334 with reverse SEQ ID NO: 7340 and any of probes SEQ ID NO: 7343-7351; Forward SEQ ID NO: 7335 and reverse SEQ ID NO: 7340 or 7341 and any of probes SEQ ID NO: 7342-7352. Other combinations of forward and reverse primers and appropriate probes can readily be determined by those skilled in the art from the above information.

Additional preferred primers and probes for SARS nucleic acid detection for diagnostic and screening are SEQ ID NOS: 7353-7362 (forward primers), SEQ ID NOS: 7363-7373 (reverse primers) and SEQ ID NOS: 7374-7385 (probes). The primers and probes are useful for detection of sequences in the 5′ UTR.

The above primers may be used in combination for amplification of SARSV nucleic acid as follows: any of forward primers SEQ ID NO: 7353-7356 with any of reverse primers SEQ ID NO: 7363-7366, 7368 and the amplified product detected (or captured) with probes SEQ ID NO: 7374; any of forward primers SEQ ID NO: 7357-7362 with any of reverse primers SEQ ID NO: 7367, 7369-7373 and the amplified products detected (or captured) with any of probes SEQ ID NO: 7375-7385. Particularly preferred combinations of forward and reverse primers and probes are: Forward primers SEQ ID NO: 7353-7356 with any of reverse primers SEQ ID NO: 7363-7366 and probes SEQ ID NO: 7374; forward primers SEQ ID NO: 7357-7358 with reverse primers SEQ ID NO: 7367, 7369 and probes SEQ ID NO: 7375 or 7376; Forward primers SEQ ID NO: 7357-7359 with reverse primers SEQ ID NO: 7367, 7369 or 7370 and probe SEQ ID NO: 7375 or 7376. More preferred are combinations of SEQ ID NO: 7353 or 7354 with SEQ ID NO: 7363 or 7364 and probe SEQ ID NO: 7374. Other combinations of forward and reverse primers and appropriate probes can readily be determined by those skilled in the art from the above information. A particularly conserved octanucleotide sequence (SEQ ID NO: 7386) occurs in the 3′UTR of SARS (approximately 70-80 bases from the 3′ end) and of several other Coronaviruses that may be particularly useful in identifying SARSV. Primers including in this region are preferably combined with reverse primers from regions of sequence that are more specific for SARS.

In addition to the above, the intergenic sequence (IS) that is characteristic of Coronavirus has been identified in SARSV (see above). The IS minimally comprises the sequence ACGAAC (SEQ ID NO: 7293) which occurs upstream of each open reading frame (ORF) in the viral genome. The 5′UTR which includes the IS is spliced onto the 5′ end of each viral mRNA at or adjacent to the site of the IS. Thus, primers comprising the IS or its complement are useful for amplifying viral nucleic acids, including cDNA made from the viral RNAs. The invention thus comprises a set of primers in which one primer comprises ACGAAC (SEQ ID NO: 7293) or its complement (SEQ ID NO: 7387) and one primer comprises any appropriate sequence from the SARS genome, or a complementary sequence. Useful probes for detecting and/or capturing the viral RNAs or cDNA made from the viral RNAs may also comprise the IS sequence, or its complement, described above.

One set of primers for amplification of SARS sequences, particularly by RT-PCR, uses SEQ ID NOs 6562, 6563, 6564 and 6565. Of these, 6562 & 6564 are sense primers and 6563 & 6565 are antisense primers. Primers SEQ ID NOS: 6562 & 6565 may be used in a first amplification, with a second nested amplification being performed using primers SEQ ID NOS: 6563 & 6564. In some embodiments of the invention, these four primers are excluded.

One kit for amplification and detection of SARS sequences, particularly by RT-PCR, uses SEQ ID NOs 6567 & 6568 as primers, and SEQ ID NO 6566 as a probe (typically labeled e.g. with TAMRA and/or FAM) for the amplified sequence. In some embodiments of the invention, these primers and probe are excluded.

One kit for amplification and detection of SARS sequences, particularly by RT-PCR, uses SEQ if NOs 7395 & 6568 as primers, and SEQ ID NO 6566 as a probe (typically labeled e.g. with TAMRA and/or FAM) for the amplified sequence. In some embodiments of the invention, these primers and probe are excluded.

One kit for amplification of SARS sequences, particularly the nucleocapsid gene, uses SEQ ID NOs 6560 & 6561 as primers. In some embodiments of the invention, these primers are excluded.

One kit for amplification of SARS sequences uses SEQ ID NOs 6496, 6497, 6562, 6563, 6564 & 6565 as primers. In some embodiments of the invention, these primers are excluded.

One kit for amplification of SARS sequences uses SEQ ID NOs 6562, 6563, 6564 & 6565 as primers. In some embodiments of the invention, these primers are excluded.

One kit for amplification of SARS sequences uses SEQ ID NOs 6500, 6501, 6502 & 6503 as primers. In some embodiments of the invention, these primers are excluded.

One kit for amplification of SARS sequences uses SEQ ID NOs 6496, 6497, 6500, 6501, 6502, 6503, 6562, 6563, 6564 & 6565 as primers. In some embodiments of the invention, these primers are excluded.

One kit for amplification and detection of SARS sequences, particularly by realtime (e.g. TaqMan™) PCR, uses SEQ ID NOs 6567 & 6568 as primers, and SEQ ID NO 6566 as a probe (typically labeled e.g. with TAMRA and/or FAM) for the amplified sequence. In some embodiments of the invention, these primers and probe are excluded.

One kit for amplification and detection of SARS sequences, particularly by realtime (e.g. TaqMan™) PCR, uses SEQ ID NOs 7395 & 6568 as primers, and SEQ ID NO 6566 as a probe (typically labeled e.g. with TAMRA and/or FAM) for the amplified sequence. In some embodiments of the invention, these primers and probe are excluded.

One kit for amplification and detection of SARS sequences uses SEQ ID NOs 6562, 6565 and 6568 as primers, and SEQ ID NOs 7396 and 7397 as probes (typically labeled e.g. with TAMRA and/or FAM) for the amplified sequence. In some embodiments of the invention, these primers and probe are excluded.

One kit for amplification and detection of SARS sequences uses an oligonucleotide comprising SEQ ID NO: 9780 as a forward primer, an oligonucleotide comprising SEQ ID NO: 9781 as a reverse primer, and an oligonucleotide comprising SEQ ID NO: 9782 as a probe.

Preferred sequences for use with RT-PCR and LightCycler analysis include SEQ ID NOs 6562, 6568, 6565, 7396 & 7397. In some embodiments of the invention, these primers and probe are excluded.

The preparation of the oligomers is by means known in the art, including, for example, by methods which include excision, transcription, or chemical synthesis. The target sequences and/or regions of the genome which are selected to which the targeting polynucleotides of the oligomers are complementary depend upon the purpose. For example, if the goal is to screen for the presence of SARSV in biological samples (e.g. blood, respiratory material, liver, lung), the preferred oligomers would be used as probes and/or primers, and would hybridize to conserved regions of the SARSV genome. Some of the conserved regions of the SARSV genome to which the oligomers may bind are described herein, for example, 5′UTR and 3′UTR.

In the basic nucleic acid hybridization assay, single-stranded analyte nucleic acid (either DNA or RNA) is hybridized to a nucleic acid probe, and resulting duplexes are detected. The probes for SARSV polynucleotides (natural or derived) are a length which allows the detection of unique viral sequences by hybridization. While 6-8 nucleotides may be a workable length, sequences of 10-12 nucleotides are preferred, and about 13, 14, 15, 16, 17, 18, 19, 20, or 21 or more nucleotides or more appears optimal. Preferably, these sequences will derive from regions which lack heterogeneity. These probes can be prepared using routine methods, including automated oligonucleotide synthetic methods. Among useful probes, for example, are those derived from less conserved regions of the SARSV genome. Regions of the genome that are typically less conserved can be readily ascertained from the sequence alignments provided herein, as well as by any other well known techniques. A complement to any unique portion of the SARSV genome will be satisfactory. For use as probes, complete complementarity is desirable, though it may be unnecessary as the length of the fragment is increased.

For use of such probes as agents to detect the presence of SARSV polynucleotides (for example in screening for contaminated blood or for diagnosing infected individuals), the biological sample to be analyzed, such as, without limitation, blood, serum, lung, liver, mucous, kidney, saliva, or sputum, may be treated, if desired, to extract the nucleic acids contained therein. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation. In order to form hybrid duplexes with the targeting sequence of the probe, the targeted region of the analyte nucleic acid must be in single stranded form. Where the sequence is naturally present in single stranded form, denaturation will not be required. However, where the sequence is present in double stranded form, the sequence will be denatured. Denaturation can be carried out by various techniques known in the art. Subsequent to denaturation, the analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte, and the resulting duplexes containing the probe(s) are detected.

Detection of the resulting duplex, if any, is usually accomplished by the use of labeled probes; alternatively, the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly. Suitable labels, and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation or kinasing), biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly triggered dioxetanes), enzymes, antibodies, and the like.

The region of the probes which are used to bind to the analyte can be made completely complementary to the SARSV genome. Therefore, usually high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency should only be used if the probes are complementary to regions of the viral genome which lack heterogeneity. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide. These factors are outlined in, for example, Maniatis T. (1982).

Variations of this basic scheme which are known in the art, including those which facilitate separation of the duplexes to be detected from extraneous materials and/or which amplify the signal from the labeled moiety, may also be used. A number of these variations are reviewed in, for example: Matthews & Kricka (1988), Analytical Biochemistry 169:1; Landegren et al. (1988), Science 242:229; and Mittlin (1989), Clinical Chem. 35:1819. These and the following publications describing assay formats are hereby incorporated by reference herein. Probes suitable for detecting SARSV in these assays are comprised of sequences which hybridize with target SARSV polynucleotide sequences to form duplexes with the analyte strand, wherein the duplexes are of sufficient stability for detection in the specified assay system.

A suitable variation is, for example, one which is described in U.S. Pat. No. 4,868,105, issued Sep. 9, 1989, and in EPO Publication No. 225,807 (published Jun. 16, 1987). These publications describe a solution phase nucleic acid hybridization assay in which the analyte nucleic acid is hybridized to a labeling probe set and to a capturing probe set. The probe-analyte complex is coupled by hybridization with a solid-supported capture probe that is complementary to the capture probe set. This permits the analyte nucleic acid to be removed from solution as a solid phase complex. Having the analyte in the form of a solid phase complex facilitates subsequent separation steps in the assay. The labeling probe set is complementary to a labeled probe that is bound through hybridization to the solid phase/analyte complex.

The polymerase chain reaction (PCR) is a technique for amplifying a desired nucleic acid sequence (target) contained in a nucleic acid or mixture thereof. In PCR, a pair of primers are employed in excess to hybridize to the complementary strands of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers then are hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules. PCR is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference.

The Ligase Chain Reaction (LCR) is an alternate method for nucleic acid amplification. In LCR, probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand, and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5′ phosphate-3′ hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in EP-A-320 308 to K. Backman published Jun. 16, 1989 and EP-A-0439182 to K. Backman et al., published Jul. 31, 1991, both of which are incorporated herein by reference.

For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, which is incorporated herein by reference; or reverse transcribe mRNA into cDNA followed by asymmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall et al., PCR Methods and Applications 4:80-84 (1994), which also is incorporated herein by reference.

TMA is described in detail in, e.g., U.S. Pat. No. 5,399,491, the disclosure of which is incorporated herein by reference in its entirety. In one example of a typical assay, an isolated nucleic acid sample, suspected of containing a SARSV target sequence, is mixed with a buffer concentrate containing the buffer, salts, magnesium, nucleotide triphosphates, primers, dithiothreitol, and spermidine. The reaction is optionally incubated at about 100° C. for approximately two minutes to denature any secondary structure. After cooling to room temperature, reverse transcriptase, RNA polymerase, and RNAse H are added and the mixture is incubated for two to four hours at 37° C. The reaction can then be assayed by denaturing the product, adding a probe solution, incubating 20 minutes at 60° C., adding a solution to selectively hydrolyze the unhybridized probe, incubating the reaction six minutes at 60° C., and measuring the remaining chemiluminescence in a luminometer.

Generally, TMA includes the following steps: (a) isolating nucleic acid, including RNA, from the biological sample of interest suspected of being infected with SARSV; and (b) combining into a reaction mixture (i) the isolated nucleic acid, (ii) first and second oligonucleotide primers, the first primer having a complexing sequence sufficiently complementary to the 3′ terminal portion of an RNA target sequence, if present (for example the (+) strand), to complex therewith, and the second primer having a complexing sequence sufficiently complementary to the 3′ terminal portion of the target sequence of its complement (for example, the (−) strand) to complex therewith, wherein the first oligonucleotide further comprises a sequence 5′ to the complexing sequence which includes a promoter, (iii) a reverse transcriptase or RNA and DNA dependent DNA polymerases, (iv) an enzyme activity which selectively degrades the RNA strand of an RNA-DNA complex (such as an RNAse H) and (v) an RNA polymerase which recognizes the promoter.

The components of the reaction mixture may be combined stepwise or at once. The reaction mixture is incubated under conditions whereby an oligonucleotide/target sequence is formed, including DNA priming and nucleic acid synthesizing conditions (including ribonucleotide triphosphates and deoxyribonucleotide triphosphates) for a period of time sufficient to provide multiple copies of the target sequence. The reaction advantageously takes place under conditions suitable for maintaining the stability of reaction components such as the component enzymes and without requiring modification or manipulation of reaction conditions during the course of the amplification reaction. Accordingly, the reaction may take place under conditions that are substantially isothermal and include substantially constant ionic strength and pH. The reaction conveniently does not require a denaturation step to separate the RNA-DNA complex produced by the first DNA extension reaction.

Suitable DNA polymerases include reverse transcriptases, such as avian myeloblastosis virus (AMV) reverse transcriptase (available from, e.g., Seikagaku America, Inc.) and Moloney murine leukemia virus (MMLV) reverse transcriptase (available from, e.g., Bethesda Research Laboratories).

Promoters or promoter sequences suitable for incorporation in the primers are nucleic acid sequences (either naturally occurring, produced synthetically or a product of a restriction digest) that are specifically recognized by an RNA polymerase that recognizes and binds to that sequence and initiates the process of transcription whereby RNA transcripts are produced. The sequence may optionally include nucleotide bases extending beyond the actual recognition site for the RNA polymerase which may impart added stability or susceptibility to degradation processes or increased transcription efficiency. Examples of useful promoters include those which are recognized by certain bacteriophage polymerases such as those from bacteriophage T3, T7 or SP6, or a promoter from E. coli. These RNA polymerases are readily available from commercial sources, such as New England Biolabs and Epicentre.

Some of the reverse transcriptases suitable for use in the methods herein have an RNAse H activity, such as AMV reverse transcriptase. It may, however, be preferable to add exogenous RNAse H, such as E. coli RNAse H, even when AMV reverse transcriptase is used. RNAse H is readily available from, e.g., Bethesda Research Laboratories.

The RNA transcripts produced by these methods may serve as templates to produce additional copies of the target sequence through the above-described mechanisms. The system is autocatalytic and amplification occurs autocatalytically without the need for repeatedly modifying or changing reaction conditions such as temperature, pH, ionic strength or the like.

Detection may be done using a wide variety of methods, including direct sequencing, hybridization with sequence-specific oligomers, gel electrophoresis and mass spectrometry. these methods can use heterogeneous or homogeneous formats, isotopic or nonisotopic labels, as well as no labels at all.

Suitable labeling moieties for attachment to primers and/or to probes used in methods of the invention include, but are not limited to: 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H), 9′-(9H)xanthene)-5-carboxylic acid,3′,6′-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein ([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloylfluoresceinyl)-6-carboxylic acid]); 6-Hexachloro-Fluorescein ([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5-Tetrachloro-Fluorescein ([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-Tetrachloro-Fluorescein ([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-6-carboxylic acid]); tetramethylrhodamines (TAMRA), including (i) 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium, 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethylamino) and (ii) 6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid); Cy5 (Indodicarbocyanine-5); Cy3 (Indodicarbocyanine-3); and BODIPY™ FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid). Labelling of probes with both FAM (e.g. at 5′) and TAMRA (e.g. at 3′) is preferred.

Nucleic acids of the invention may be used in solution or may be bound to a solid matrix or support e.g. in the format of a DNA array,

As is readily apparent, design of the assays described herein are subject to a great deal of variation, and many formats are known in the art. The above descriptions are merely provided as guidance and one of skill in the art can readily modify the described protocols, using techniques well known in the art.

One 302nt amplicon of the SARS virus is known as “BNI-1” (SEQ ID NO: 9927). It was sequenced at the Bernhard Nocht Institute, Hamburg, Germany. In April 2003 the BNI-1 sequence was published on the WHO website (http://www.who.int/csr/sars/primers/en/) and in Dorsten et al., “Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome”, New England Journal of Medicine, published online at http://www.nejm.org. Both references are incorporated herein by reference in their entirety. Some embodiments of the invention do not encompass a nucleic acid consisting of SEQ ID NO: 9927. Some other embodiments of the invention do not encompass a nucleic acid comprising SEQ ID NO: 9927. Some embodiments of the invention do not encompass a polypeptide consisting of any one of SEQ ID NO^(S): 9928 to 9959. Some other embodiments of the invention do not encompass a nucleic acid comprising any one of SEQ ID NOs: 9928 to 9959. Some embodiments of the invention are not subject to these exclusions.

Immunoassays

The present invention utilizes various immunoassay techniques for identifying individuals exposed to SARSV and/or biological samples containing SARSV antigens or antibodies to SARSV.

Immunoassay Formats

The SARSV antigens may be employed in virtually any assay format that employs a known antigen to detect antibodies. A common feature of all of these assays is that the antigen is contacted with biological sample suspected of containing SARSV antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strength using an excess of antigen. The incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen. Alternatively, anti-SARSV antibodies may be employed to detect the presence of SARSV antigens in a biological sample. Combination antigen/antibody assays are also contemplated; for example, as described for HCV detection in US patent 6,630,298.

Design of the immunoassays is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

The immunoassay may be, without limitation, in a heterogeneous or in a homogeneous format, and of a standard or competitive type. In a heterogeneous format, the polypeptide is typically bound to a solid matrix or support to facilitate separation of the sample from the polypeptide after incubation. Examples of solid supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride, diazotized paper, nylon membranes, microchips, high or low density biochips, recombinant immunoassays (RIBA), microfluidity devices, micromagnetic beads, activated beads, and Protein A beads. For example, Dynatech Immunlon or Immunlon 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous format. The solid support containing the antigenic polypeptides is typically washed after separating it from the test sample, and prior to detection of bound antibodies. Both standard and competitive formats are known in the art.

In a homogenous format, the test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes which are formed. Both standard and competitive formats for these assays are known in the art.

In a standard format, the amount of SARSV antibodies in the antibody-antigen complexes is directly monitored. This may be accomplished by determining whether labeled anti-xenogeneic (e.g., anti-human) antibodies which recognize an epitope on anti-SARSV antibodies will bind due to complex formation. In a competitive format, the amount of SARSV antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) in the complex.

Complexes formed comprising anti-SARSV antibody (or in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled SARSV antibodies in the complex may be detected using a conjugate of antixenogeneic Ig complexed with a label, (e.g., an enzyme label).

In an immunoprecipitation or agglutination assay format the reaction between the SARSV antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-SARSV antibody is present in the test specimen, no visible precipitate is formed.

There are at least three specific types of particle agglutination (PA) assays. These assays are used for the detection of antibodies to various antigens when coated to a support. One type of this assay is the hemagglutination assay using red blood cells (RBCs) that are sensitized by passively adsorbing antigen (or antibody) to the RBC. The addition of specific antigen antibodies present in the body component, if any, causes the RBCs coated with the purified antigen to agglutinate.

To eliminate potential non-specific reactions in the hemagglutination assay, two artificial carriers may be used instead of RBC in the PA. The most common of these are latex particles. However, gelatin particles may also be used. The assays utilizing either of these carriers are based on passive agglutination of the particles coated with purified antigens.

The SARSV antigens will typically be packaged in the form of a kit for use in these immunoassays. The kit will normally contain in separate containers the native SARSV antigen, control antibody formulations (positive and/or negative), labeled antibody when the assay format requires same and signal generating reagents (e.g., enzyme substrate) if the label does not generate a signal directly. The native SARSV antigen may be already bound to a solid matrix or separate with reagents for binding it to the matrix. Instructions (e.g., written, tape, CD-ROM, etc.) for carrying out the assay usually will be included in the kit.

Immunoassays that utilize the native SARSV antigen are additionally useful in screening blood for the preparation of a supply from which potentially infective SARSV is lacking. The method for the preparation of the blood supply comprises the following steps. Reacting a body component, preferably blood or a blood component, from the individual donating blood with native SARSV antigen to allow an immunological reaction between SARSV antibodies, if any, and the SARSV antigen. Detecting whether anti-SARSV antibody-SARSV antigen complexes are formed as a result of the reacting. Blood contributed to the blood supply is from donors that do not exhibit antibodies to the native SARSV antigens.

Production of Antibodies

As explained above, the assay may utilize various antibodies which may be bound to a solid support, and that detect antigen or antigen/antibody complexes formed when SARSV infection is present in the sample. These antibodies may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, or may be hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab′)₂ fragments, F(ab) fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragment constructs, minibodies, or functional fragments thereof which bind to the antigen in question.

Antibodies are produced using techniques well known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745. For example, polyclonal antibodies are generated by immunizing a suitable animal, such as a mouse, rat, rabbit, sheep or goat, with an antigen of interest. In order to enhance immunogenicity, the antigen can be linked to a carrier prior to immunization. Such carriers are well known to those of ordinary skill in the art. Immunization is generally performed by mixing or emulsifying the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). The animal is generally boosted 2-6 weeks later with one or more injections of the antigen in saline, preferably using Freund's incomplete adjuvant. Antibodies may also be generated by in vitro immunization, using methods known in the art. Polyclonal antiserum is then obtained from the immunized animal.

Monoclonal antibodies are generally prepared using the method of Kohler & Milstein (1975) Nature 256:495-497, or a modification thereof, as described above.

As explained above, antibody fragments which retain the ability to recognize the antigen of interest, will also find use in the subject immunoassays. A number of antibody fragments are known in the art which comprise antigen-binding sites capable of exhibiting immunological binding properties of an intact antibody molecule. For example, functional antibody fragments can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g., pepsin, to produce F(ab′)₂ fragments. These fragments will contain two antigen binding sites, but lack a portion of the constant region from each of the heavy chains. Similarly, if desired, Fab fragments, comprising a single antigen binding site, can be produced, e.g., by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced, using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as Fv. See, e.g., Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single-chain Fv (“sFv” or “scFv”) polypeptide is a covalently linked V_(H)-V_(L) heterodimer which is expressed from a gene fusion including V_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883. A number of methods have been described to discern and develop chemical structures (linkers) for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. The sFv molecules may be produced using methods described in the art. See, e.g., Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85:5879-5883; U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. Design criteria include determining the appropriate length to span the distance between the C-terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Such methods have been described in the art. See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,946,778. Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility.

“Mini-antibodies” or “minibodies” will also find use with the present invention. Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region. Pack et al. (1992) Biochem 31:1579-1584. The oligomerization domain comprises self-associating a-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds. The oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein. Generally, minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J. Immunology 149B: 120-126.

Production of SARS Antigens

The SARSV antigens used in the present invention are generally produced recombinantly. Thus, polynucleotides encoding SARSV antigens for use with the present invention can be made using standard techniques of molecular biology. For example, polynucleotide sequences coding for the above-described molecules can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells expressing the gene, or by deriving the gene from a vector known to include the same. Furthermore, the desired gene can be isolated directly from viral nucleic acid molecules, using techniques described in the art, such as those described for HCV in Houghton et al., U.S. Pat. No. 5,350,671. The gene encoding the antigen of interest can also be produced synthetically, rather than cloned. The molecules can be designed with appropriate codons for the particular sequence (preferably optimum codons for the expression host of choice). The complete sequence is then assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; and Jay et al. (1984) J. Biol. Chem. 259:6311.

Thus, particular nucleotide sequences can be obtained from vectors harboring the desired sequences or synthesized completely or in part using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate. See, e.g., Sambrook, supra. In particular, one method of obtaining nucleotide sequences encoding the desired sequences is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR. See, e.g., Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088. Additionally, oligonucleotide directed synthesis (Jones et al. (1986) Nature 54:75-82), oligonucleotide directed mutagenesis of pre-existing nucleotide regions (Riechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al. (1988) Science 239:1534-1536), and enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase (Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033) can be used under the invention to provide molecules having altered or enhanced antigen-binding capabilities, and/or reduced immunogenicity.

Once coding sequences have been prepared or isolated, such sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Suitable vectors include, but are not limited to, plasmids, phages, transposons, cosmids, chromosomes (including artificial chromosomes, such as BACs or YACs) or viruses which are capable of replication when associated with the proper control elements.

The coding sequence is then placed under the control of suitable control elements, depending on the system to be used for expression. Thus, the coding sequence can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence of interest is transcribed into RNA by a suitable transformant. The coding sequence may or may not contain a signal peptide or leader sequence which can later be removed by the host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of the expression of the sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector. For example, enhancer elements may be used herein to increase expression levels of the constructs. Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO J 4:761), the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived from human CMV (Boshart et al. (1985) Cell 41:521), such as elements included in the CMV intron A sequence (U.S. Pat. No. 5,688,688). The expression cassette may further include an origin of replication for autonomous replication in a suitable host cell, one or more selectable markers, one or more restriction sites, a potential for high copy number and a strong promoter.

An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the “control” of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification of the sequences encoding the molecule of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it can be attached to the control sequences in the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

As explained above, it may also be desirable to produce mutants or analogs of the antigen of interest. Methods for doing so are described in, e.g., Dasmahapatra et al., U.S. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. No. 5,990,276. Mutants or analogs of SARSV proteins for use in the subject assays may be prepared by the deletion of a portion of the sequence encoding the polypeptide of interest, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, and the like, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. (1987) BioTechniques 5:786; Zoller & Smith (1983) Methods Enzymol. 100:468; Dalbie-McFarland et al. (1982) Proc. Natl. Acad. Sci USA 79:6409.

The molecules can be expressed in a wide variety of systems, including insect, mammalian, bacterial, viral and yeast expression systems, all well known in the art.

For example, insect cell expression systems, such as baculovirus systems, are known to those of skill in the art and described in, e.g., Summers & Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). Similarly, bacterial and mammalian cell expression systems are well known in the art and described in, e.g., Sambrook et al., supra. Yeast expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

A number of appropriate host cells for use with the above systems are also known. For example, mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human embryonic kidney cells, human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa califormica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Nucleic acid molecules comprising nucleotide sequences of interest can be stably integrated into a host cell genome or maintained on a stable episomal element in a suitable host cell using various gene delivery techniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346.

Depending on the expression system and host selected, the molecules are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein is expressed. The expressed protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the product can be purified directly from the media. If it is not secreted, it can be isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.

EXAMPLE

For useful expression of SARSV antigens in Saccharomyces cerevisiae and Pichia pastoris, insect cells, and mammalian cells, the following domains are cloned into expression vectors as listed in the Table below. The nt sequence numbers are from the SARSV sequence of SEQ ID NO: 1.

-   -   RNA polymerase 1a: SARS nt 250-13398     -   RNA polymerase 1b: SARS nt 13399-21470     -   ORFns.envelope (homologous to ns2, hemagglutinin-esterase         envelope glycoprotein, and spike glycoprotein): SARS nt         21477-25244     -   Membrane: SARS nt 27849-28103     -   Nucleocapsid: SARS nt 28105-29373

A combination of PCR and synthetic oligos is used to create the above domains with restriction sites tailored to the following expression vectors: Restriction ends Vector Promoter Expression host HindIII/SalI pBS24.1 ADH2/GAPDH AD3/Saccharomyces EcoRI/Sal\\\ pBS24.1 ADH2/GAPDH/SOD fusion AD3/Saccharomyces XbaI/SalI pAO815 AOXI GS115/Pichia pastoris HVK-293/Transient EcoRI/BamHI pCMVkm2 CMVp/Enhancer/IntronA transfection EcoRI/XmaI pCMVIII CMVp/Enhancer/IntronA CHO stable cell line Cell lines employed by Chiron NheI/SalI pBluBac4.5 Polyhedrin include: Sf9, Sf21, Tn5 IV. Treatment of SARS Infection With RNAi

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., Nature 391, 806-811(1998)). RNAi most likely involves mRNA degradation, resulting in sequence-specific, post-transcriptional gene silencing in many organisms. RNAi is a post-transcriptional process triggered by the introduction of double-stranded RNA which leads to gene silencing in a sequence-specific manner. RNAi has been reported to occur naturally in organisms as diverse as nematodes, trypanosmes, plants and fungi. It most likely serves to protect organisms from viruses, modulate transposon activity and eliminate aberrant transcription products.

The first evidence that dsRNA could achieve efficient gene silencing through RNAi came from studies on the nematode Caenorhabditis elegans (Fire et al. (1998) Nature, 391:806-811 and U.S. Pat. No. 6,506,559). Later studies in the fruit fly Drosophila melanogaster demonstrated that RNAi is a two-step mechanism (Elbashir et al. (2001) Genes Dev., 15(2): 188-200). First, long dsRNAs are cleaved by an enzyme known as Dicer in 21-23 nucleotides (nt) fragments, called small interfering RNAs (siRNAs). Then, siRNAs associate with a ribonuclease complex (termed RISC for RNA Induced Silencing Complex) which target this complex to complementary mRNAs. RISC then cleaves the targeted mRNAs opposite the complementary siRNA, which makes the mRNA susceptible to other RNA degradation pathways.

RNAi is the phenomenon where dsRNA corresponding to a targeted DNA or RNA sequence can suppress or silence gene expression. Even though dsRNA can mediate gene-specific interference in mammalian cells in some circumstances (Wianny & Zernicka-Goetz (2000) Nature Cell Biol. 2:70-75; Svoboda et al. (2000) Development 17:4147-4156) the use of RNAi in mammalian somatic cells is often limited due to the dsRNA triggering dsRNA-dependent protein kinase (PKR) which in turn inactivates translation factor eIF2a and causes a generalized suppression of protein synthesis and often times apoptosis (Gil & Esteban (2000) Apoptosis 5:107-114).

Recently, gene-specific suppression using siRNA of approximately 21 or 22 base pairs in length, corresponding to targeted RNA or DNA sequences, were shown to disrupt the expression of these targeted sequences in mammalian cells (Elbashir, S. M., et al., Nature 411: 494-498 (2001)). However, it is not clear that all RNA or DNA sequences of a mammalian cell's genome are susceptible to siRNA. It is also uncertain that every mammalian cell type possesses the necessary machinery for effecting gene-specific suppression using siRNA. Further, siRNA is of limited use for at least two reasons: the transient nature of the suppression effect seen in cells where the siRNA has been administered; and in some instances the necessity for chemical synthesis of siRNAs before their use (Tuschl T., Nature Biotechnol., 20: 446-448 (2002)). Also the instability of these short, synthetic RNAs makes it presents problems for any long term use of these siRNAs a pharmaceutical.

To overcome this limitation, the present invention provides a modified siRNA with increased stability against nuclease degradation while still maintaining its ability to inhibit viral replication via RNA interference. Such modification to the ribonucleotides in the siRNAs, adds a chemical group via chemical synthesis or in vitro transcription or longer modified RNAs can be prepared by either of these methods and cut into siRNAs using Dicer.

Although other methods for gene-specific suppression have utilized chemically-modified nucleic acids, such as antisense and ribozyme technology, such modification destroys critical enzymatic activities necessary for the function of these technologies. In regard to antisense technology, modification of the ribonucleotides destroys RNaseH activity, whereas such modification abolishes the catalytic activity of ribozymes.

The present invention provides a double-stranded RNA (dsRNA) molecule modified for protection against nuclease degradation with a length from about 10 to about 30 nucleotides which is able to inactivate a virus in a mammalian cell. The invention also provides a method of inactivating a virus by administering modified small interfering RNAs (siRNAs) that are modified so that they are nuclease or RNase resistant and retain the biological activity of being able to inhibit viral replication by targeting a RNA sequence in a virus.

The invention is further directed to a method of making modified siRNAs that target a RNA sequence in a virus comprising preparing a modified-double stranded RNA (dsRNA) fragment containing at least one modified ribonucleotide in at least one strand that spans the genome of the virus; and cleaving the modified-dsRNA fragments with recombinant human Dicer resulting in more than one modified siRNA.

The present invention provides a modified dsRNA molecule of from about 10 to about 30 nucleotides which mediates targeted RNA interference in hepatic or SARS-infected cells.

As used herein RNA interference, or RNAi, is used to mean sequence-specific, or gene specific, suppression of gene expression (protein synthesis), without causing a generalized suppression of protein synthesis in cells harboring the siRNA. The invention is not limited to a particular theory of the mechanism of action of RNAi. For example, RNAi may involve degradation of messenger RNA (mRNA) in an RNA-induced silencing complex (RISC), preventing translation of the transcribed mRNA, or it may involve the methylation of genomic DNA, shunting transcription of the gene. The lack of gene expression caused by RNAi may be transient, lasting a short period of time, or it may be stable, or permanent, lasting an indefinite period of time.

The term RNA is meant as is recognized in the art. Further, as used herein, RNA is used to mean double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) or a dsRNA with a single-stranded overhang. dsRNAs-within the meaning of the present invention includes short interfering RNA (siRNA), micro RNA (miRNA) and small hairpin RNA (shRNA), Additionally, RNA is also used to mean messenger RNA (mRNA), transfer RNA (tRNA) or ribosomal RNA (rRNA).

The present invention is directed to small interfering RNA (siRNA) which have been chemically modified to confer increased stability against nuclease degradation yet these siRNAs are still able to bind to target RNAs, that may be present in a cells. In the case where the target RNA is a virus specific RNA, the modified siRNAs are able to bind to the virus specific RNAs and inactivate the virus. A modified siRNA of the present invention comprises a modified ribonucleotide, wherein the siRNA is resistant to enzymatic degradation, such as RNase degradation, and yet retains the ability to inhibit viral replication. The modified siRNA is more specifically modified at the 2′ position of the ribose in the siRNA. The modification is at the 2′ position of at least one ribonucleotide of said siRNA. Attachment of receptor-binding ligands to siRNA molecules can be used to target the siRNA to a desired cell type. For example, attachment of cholesterol at the 5′-end or 3′-end of the siRNA molecule, to give a cholesteryl siRNA, can enhance targeting to hepatocytes. Other ligands for receptor mediated siRNA targeting to liver include HBV surface antigen, LDL, and others.

More specifically, the siRNA is modified at at least one pyrimidine, at least one purine or a combination thereof. However, generally all pyrimidines, or all purines or a combination of all pyrimidines and all purines of the siRNA are modified. More preferably, the pyrimidines are modified and these pyrimidines are cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof. It also is contemplated to modify the selected ribonucleotides in at least one strand of the siRNA or the ribonucleotides in both strands of the siRNA are modified.

The nucleotides containing pyrimidine bases found in RNA (cytidine and uridine) can be chemically modified by adding any molecule that inhibits RNA degradation or breakdown to the 2′ position of the ribose molecule. The 2′-modified pyrimidine nucleotide can be formed using a number of different methods. The 2′ modification confers increased stability to the siRNA by making the siRNA impervious or resistant to nuclease activity. Thus, the 2′ modified siRNA has a longer serum half-life and is resistant to degradation compared to unmodified siRNA. The siRNA also may be modified completely or partially.

Regarding chemical modification of siRNAs, a molecule from the halide chemical group is preferably added to the ribonucleotide of the siRNA. Within the halides, fluorine is the preferred molecule but other chemical molecules, in addition to fluoro-, such as methyl-, methoxyethyl- and propyl-modifications can also we made. But the preferred modications is fluoro-modification, such as a 2′-fluoro-modication or a 2′,2′-fluoro-modification. Thus, in a preferred embodiment of the invention, the siRNA is modified by adding a fluorine molecule to the 2′ carbon of the pyrimidine ribonucleotide. The siRNA may be fluorinated completely or partially. For example, only the cytosine nucleotides need be fluorinated. Alternatively, only the uracil nucleotide need be fluorinated but both uracil and cytosine can be fluorinated. Furthermore, only one strand, either sense or antisense, of the siRNA can be fluorinated. Even partial 2′ fluorination the siRNA gives protection against nucleolytic degradation. Furthermore, it is important to note the 2′ fluorinated siRNA is not toxic to cells, an unexpected result given that fluorine chemistry usually is toxic to living organisms.

The siRNA of the present invention is designed to interact with a target nucleotide sequence. Most preferably this target nucleotide sequence is a disease producing agent or pathogen of which one wishes to inhibit gene expression. More preferably, this target nucleotide sequence is in a virus genome, and further this virus genome is from a RNA virus or a DNA virus is selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus. The most preferred virus is SARS virus.

Modfied siRNA may be prepared in a number of ways, such as by chemical synthesis, T7 polymerase transcription, or by treating modified long double stranded RNA (dsRNA) prepared by one of the two previous methods with Dicer enzyme. Dicer enzyme can be used to cleave dsRNA that is about 500 base pairs to about 1000 base pairs in size, to created mixed populations of dsRNA from about 21 to about 23 base pairs in length. Furthermore, an unexpected result of using the Dicer enzyme method is that Dicer enzyme will cleave modified strands of dsRNA, such as 2′ fluorinated modified dsRNA. Before development of this method, it was previously thought that Dicer would not be able to cleave modified siRNA. The Dicer method can be carried out using the Dicer siRNA Generation Kit available from Gene Therapy Systems, San Diego, Calif.

As used herein, small interfering RNA (siRNA) is defined as double- or single-stranded RNA of from about 10 to about 30 nucleotides in length, more preferably 12-28 nucleotides, more preferably 15-25 nucleotides, even more preferably 19-23 nucleotides and most preferably 21-23 nucleotides. The length of a siRNA as used herein, is determined by the length of one of the strands of the RNA. For example, a siRNA that is described as 21 nucleotides long (a 21-mer) may comprise two opposite strands of RNA which anneal together for 19 contiguous base pairings. The two remaining nucleotides on one end of the molecule would not anneal to the opposite strand, thus creating an “overhang”. The overhang can be at the 5′ or the 3′ end of the dsRNA. Preferably, the overhang is at the 3′ end of the RNA strand. The length of a double-stranded RNA where the two opposite strands are not the same length will be designated by the longer of the two strands. For example, a dsRNA comprising one strand which is 21 nucleotides long and anneals to an opposite strand that is 20 nucleotides long, will be considered, as used herein, a 21-mer.

Preferably, the siRNA of the present invention will comprise a 3′ overhang of about 2 to 4 bases. More preferably, the 3′ overhang is 2 nucleotides long. Even more preferably, the 2 nucleotides comprising the 3′ overhang are uridine (U).

In one embodiment, the invention provides an RNA molecule comprising a nucleotide sequence at least 80% identical to the nucleotide sequence of the target agent or virus. Preferably, the RNA molecule of the present invention is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of the target agent or virus.

As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97% 98%, 99% or 100% identical to the nucleotide sequence of the target agent or virus can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith & Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

The present invention provides a method of inactivating a target agent or preferably a virus in a patient comprising administering to the patient a modified siRNA in an effective amount to inactivate the targeted agent or virus. RNA interference towards a targeted DNA segment in a cell can be achieved by administering a dsRNA molecule or siRNA to the cells, wherein the nucleotide sequence of the dsRNA molecule corresponds to the nucleotide sequence of the targeted DNA segment. Preferably, the RNA molecule used to induce targeted RNAi is siRNA.

Gene suppression, targeted suppression, sequence-specific suppression, targeted RNAi or sequence-specific RNAi are used interchangeably herein. Furthermore, sequence-specific suppression, as used herein, is determined by separately assaying the levels of the protein targeted for suppression in cells containing the siRNA (experimental cells) and in cells not containing the identical siRNA (control cells), and comparing the two values. Furthermore, the experimental and control cells must be derived from the same source and same animal. For example, the control and experimental cells can be, but are not limited to, normal human hepatic cells as cell culture in vitro, or they can derived from a hepatocellular carcinoma. Further, the control and experimental cells used in determining the level or quantity of gene suppression must be assayed under similar, if not identical, conditions.

As used herein the phrase “targeted DNA segment” is used to mean a DNA sequence encoding, in whole or in part, an mRNA for a targeted protein, including introns or exons, where suppression is desired. DNA segment can also mean a DNA sequence that normally regulates expression of the targeted protein, including but not limited to the promoter of the targeted protein. Furthermore, the DNA segment may or may not be a part of the cell's genome or it may be extrachromosomal, such as plasmid DNA.

The present invention is further directed to inactivating a virus in a patient comprising administering to a patient a modified siRNA in an effective amount to inactivate the virus. The siRNA is preferably about 10 to about 30 nucleotides in length, more preferably 12-28 nucleotides, more preferably 15-25 nucleotides, even more preferably 19-23 nucleotides and most preferably 21-23 nucleotides. The method preferably utilizes a 2′ modified siRNA that is modified at the 2′ position of at least one ribonucleotide of said siRNA. The method utilizes a siRNA that is modified with chemical groups selected from the group consisting of fluoro-, methyl-, methoxyethyl- and propyl-modification. The fluoro-modification is preferred and either a 2′-fluoro-modication or a 2′,2′-fluoro-modification is useful in the present invention and preferred.

The modification may be at the pyrimidines, the purines or a combination thereof of the siRNA are modified. More preferably the pyrimidines are modified, such as cytosine, a derivative of cytosine, uracil, a derivative of uracil or a combination thereof. In one embodiment, at least one strand of the siRNA contains at least one modified nucleotide and in an alternate embodiment, oth strands of the siRNA contains at least one modified nucleotide.

The method is intended to target disease causing agents or pathogens, an more particularly viruses, which can be either a RNA virus or a DNA virus, which are selected from the group consisting of hepatitis C virus (HCV), hepatitis A virus, hepatitis B virus, hepatitis D virus, hepatitis E virus, Ebola virus, influenza virus, rotavirus, reovirus, retrovirus, poliovirus, human papilloma virus (HPV), metapneumovirus and coronavirus. More preferably the target virus is a SARS virus. The present method utilizes a siRNA prepared by (a) identifying a target nucleotide sequence in a virus genome, preferably SARS virus, for designing a small interfering RNA (siRNA); and (b) producing a siRNA that has been modified to contain at least one modified nucleotide. More preferably, the siRNA comprises a dsRNA molecule with a first strand ribonucleotide sequence corresponding to a nucleotide sequence corresponding to a target nucleotide sequence in said virus and a second strand comprising a ribonucleotide sequence complementary to said target nucleotide sequence, wherein said first and second strands are separate complementary strands that hybridize to each other to form said dsRNA molecule, and further wherein the first strand ribonucleotide sequence, the second strand ribonucleotide sequence or both the first and second strand ribonucletide sequences comprise at least one modified nucleotide. In this method, the target nucleotide sequence comprises a conserved nucleotide sequence necessary for SARS virus replication, and the conserved nucleotide sequence is selected from the group consisting of SEQ ID NO: 7292, SEQ ID NO: 7293, SEQ ID NO: 7294, SEQ ID NO: 7295, SEQ ID NO: 7296, SEQ ID NO: 7297, SEQ ID NO: 7298, SEQ ID NO: 7299, SEQ ID NO: 7300 and SEQ ID NO: 7301. Preferably, the nucleotide sequence is selected from the group consisting of SEQ ID NO: 7292 and SEQ ID NO: 7293. Still more preferably, the nucleotide sequence is SEQ ID NO: 7293.

The siRNA disclosed in this application may be prepared with modified ribonucleotides as described herein. Further, the modified ribonucleotide of the siRNA used in the present method is incorporated into said siRNA by chemical synthesis or enzymatic synthesis.

The siRNA disclosed in this application may or may not have a 5′ triphosphate group.

The modified siRNA is administered to a patient by a method selected from the group consisting of intravenous injection, subcutaneous injection, oral delivery, and liposome delivery. The modified siRNA accumulates in an organ, tissue or body system of the patient that are the liver, gastrointestinal tract, respiratory tract, cervix or skin.

The present invention also provides a method of inhibiting the replication of a virus, such as SARS virus, in cells positive for SARS virus comprising transfecting SARS-positive cells with a vector that directs the expression of modified siRNA which is specific for SARS. The cells are evaluated to determine if a marker in the cells has been inhibited by the modified siRNA.

The term patient, as used herein, can be an animal, preferably a mammal. More preferably the subject can be a primate, including non-human and humans. The terms subject and patient can be used interchangeably.

The treatment envisioned by the current invention can be used for subjects with a pre-existing viral infection, or for subjects pre-disposed to a SARS virus infection. Additionally, the method of the current invention can be used to correct or compensate for cellular or physiological abnormalities involved in conferring susceptibility to viral infections in patients, and/or to alleviate symptoms of a viral infection in patients, or as a preventative measure in patients.

The method of treating a patient having a viral infection involves administration of compositions to the subjects. As used herein, composition can mean a pure compound, agent or substance or a mixture of two or more compounds, agents or substances. As used herein, the term agent, substance or compound is intended to mean a protein, nucleic acid, carbohydrate, lipid, polymer or a small molecule, such as a drug.

In one embodiment of the current invention, the composition administered to the subject is a pharmaceutical composition. Further, the pharmaceutical composition can be administered orally, nasally, parenterally, intrasystemically, intraperitoneally, topically (as by drops or transdermal patch), bucally, or as an oral or nasal spray. The term “parenteral,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The pharmaceutical compositions as contemplated by the current invention may also include a pharmaceutically acceptable carrier.

By “pharmaceutically acceptable carrier” is intended, but not limited to, a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type, such as liposomes.

A pharmaceutical composition of the present invention for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The compositions of the present invention can also contain adjuvants such as, but not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of the drugs, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compounds are mixed with at least one item pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl -sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Alternatively, the composition can be pressurized and contain a compressed gas, such as nitrogen or a liquefied gas propellant. The liquefied propellant medium and indeed the total composition is preferably such that the active ingredients do not dissolve therein to any substantial extent. The pressurized composition can also contain a surface active agent. The surface active agent can be a liquid or solid non-ionic surface active agent or can be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.

The compositions of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to the compounds of the invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (see, for example, Prescott, Ed., Meth. Cell Biol. 14:33 et seq (1976)).

One of ordinary skill will appreciate that effective amounts of the agents of the invention can be determined empirically and can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form. The agents can be administered to a subject, in need of treatment of viral infection, as pharmaceutical compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents or composition of the present invention will be decided by the attending physician within the scope of sound medical judgement. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular or physiological response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.

Dosing can also be arranged in a patient specific manner to provide a predetermined concentration of the agents in the blood, as determined by techniques accepted and routine in the art. Thus patient dosaging can be adjusted to achieve regular on-going blood levels, as measured by HPLC, on the order of from 50 to 1000 ng/ml.

It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of the invention or any embodiment thereof.

The modified siRNA is prepared by custom chemical synthesis by Dharmacon, at Lafayette Colo. Each C and U within the siRNA duplex (GL2), has been substituted with 2′-F-U and 2′-F-C except for the 3′-end overhang, which was dTdT.

To test the stability of 2′ chemically modified siRNA compared to unmodified siRNA (siRNA), the following experiment is performed. 4 ngs of siRNA are added to a 20 μL volume of 80% human serum from a healthy donor. This mixture is incubated at 37° C. for various times ranging from 1 minute up to 10 days. The same process is performed for 2′ fluorine modified siRNA (2′-F siRNA). When the incubation process is finished, the mixtures are placed on ice and then immediately separated by PAGE along with a ³²P-siRNA control. The 2′ modified siRNA is stable as compared to unmodified siRNA.

V. Identification of Therapeutically Active Agents for Treatment of SARS Virus Infection

The invention provides methods for treating SARS by administering therapeutically active agents, such as small molecule compounds, to a mammal, as well as methods of identifying therapeutically active agents, such as potent small molecules, for the treatment of SARS virus infection.

In one aspect of the invention a method of identifying a therapeutically active agent is provided comprising: (a) contacting the therapeutically active agent with a cell infected with the SARS virus; (b) measuring attenuation of a SARS related enzyme.

In a more particular embodiment, the therapeutically active agent is a small molecule. In another more particular embodiment, the therapeutically active agent is a nucleoside analog (e.g. Ribavirin). In another more particular embodiment the small molecule is a SMIP or peptidic immunomodulating compound. In another more particular embodiment the therapeutically active agent is a peptoid, oligopeptide, or polypeptide. In another embodiment the SARS related enzyme is SARS protease. In another embodiment the SARS related enzyme is SARS polymerase. In still another embodiment the SARS related enzyme is a kinase. In still another embodiment, the SARS related enzyme is a protease. The furin inhibitor peptidyl chloromethylketone prevents blocks cell-cell fusion after MHV infection (de Haan et al. (2004) J Virol), which offers guidance for SARS therapy.

The invention includes a cell-based assay that can be used to screen for and identify a therapeutically active agent for the treatment of SARS virus infection. Therapeutically active agents of the invention include agents that inhibit, prevent or reduce the replication of a SARS virus. Such agents can be identified by infecting a cultured cell (such as, for example, VERO cells) with a SARS virus and evaluating the impact of potential antiviral compounds on SARS virus replication. Assays to measure the effect of a potential antiviral compound on virus replication are known in the art and may be based on a variety of parameters.

The cell-based assay may be used in a high-throughput screen to identify therapeutically active compounds from chemical libraries comprising potential antiviral compounds. Therapeutically active compounds suitable for use in the invention may inhibit any SARS viral target that is essential for viral replication in whole cells. Efficacy (the ability of a compound to inhibit or inactivate the target, be it viral or cellular, that results in the reduction of virus in the culture) of the therapeutic agent is measured by assessing the viability and/or the proliferation of surviving cells in a SARS virus infected cell culture.

A number of methods can be used to measure cell viability are known in the art, such as assays measuring cellular enzymes, proteins, nucleotide triphoshates (e.g. ATP), nucleic acids (e.g. host cell mRNA (e.g. GAPDH) or rRNA sequences) or cellular metabolites such as MTT or MTS. In addition, fluorescent (including, for example HSV paper) or non-fluorescent dyes (e.g. propidium diiodide) or labeling of DNA can be used to measure indications of cell viability and/or proliferation.

Alternatively, efficacy of a compound or sample can be determined by directly measuring the amount of virus or viral products in the culture. Methods for measuring the amount of virus, viral genome or viral products include: PCR, RT-PCR, TMA, reporter proteins with fluourescent or luminescent qualities or enzymatic functions (e.g., luciferase, alkaline phosphatase, GFP) or proteins that can be detected by antibodies (e.g. EGF) that might be incorporated into the viral genome prior to infection of the cell culture. Further, viral products such as viral proteins can be measured by ELISA or enzymatic activities. Methods for identifying viral polynucleotides, viral proteins and antibodies specific to viral proteins are discussed above.

Potential antiviral compounds are applied to the cell-based assay at a concentration of approximately 10 μM and compound classes having therapeutic effect are identified by measuring the parameter of choice (such as cell viability/proliferation or the virus or viral genome or a viral product be it viral in origin or non-virus in orgin). Once compounds are identified as having activity, they are resynthesized, and analoged. Starting with the identified compound, many analogs and new compounds are synthesized during consecutive optimization cycles of synthesis, biological profiling and modeling techniques to optimize the to the lead structure until in vivo activity is elucidated and optimized.

Cells suitable for use in the assay include the cells described above as suitable for vaccine production. Preferably, the cells are African green monkey kidney cells (Vero) cells. Human embyronic lung fibroblasts or normal human diploid fibroblasts may also be used in the invention.

In one embodiment, the invention includes a fluorescence based cytopathogenicity assay to measure the effect of a potential antiviral compound on a cell-based assay. One example of a fluorescence based cytopathogenicity assay is illustrated below.

1×10⁴ Vero cells per well of a microtiter plate (MTP) are infected with a defined amount of SARS virus selected within the following ranges for optimal MOI: 5-10, 10-25, 25-50, 50-100, 100-500, or 500-1000 PFU in a total volume of 200 μl media (M199 medium supplemented with 5% FCS, 2 mM glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin) in the presence or absence of the potential antiviral compound and incubated for at least 1, 2, 3, 4, 5, 6, or 7 days at 37° C., 5% CO₂. The wells of the MTP are washed with PBS (200 μl) and then filled with 200 μl PBS containing 10 μg/ml fluorescein diacetate. After a 45-min incubation at room temperature, fluorescence is measured at 485 nm excitation and 538 nm emission wavelengths. IC₅₀ values are determined by a nonlinear plot of antiviral activity as a function of drug concentration.

Other cell based assays are known in the art and include, among others, methods of GFP detection and Luc detection. In addition, a Promega kit is commercially available that provides additional methods of measuring cell viability, etc.

In one embodiment, the invention includes a method of measuring the efficacy of a potential antiviral compound using RT-PCR to detect the levels of SARS viral RNA in the cell based assay. Methods of using RT-PCR are known in the art. One example of such an assay is described below.

5×10⁶ Vero cells are seeded in tissue culture. Flasks containing the cells are incubated over night at 37° C., 5% CO₂. The cells are infected (m.o.i.=1) with SARS virus in the presence and absence of potential antiviral compounds. Optionally, the cells may be pretreated with the potential compound prior to infection. In either case, a suitable control cell assay is also prepared.

The RNA of infected cells is purified at 2 h (UL54), 12 h (UL8) and 16 h (UL13) after infection, (Qiagen) RNA purification (RNeasy kit; 40 μl elution) and quantified (absorption at 260 nm). The RNA (2 μg) is reverse transcribed with a specific primer (2 pmol, using one of the primer pairs described herein) into cDNA according to the Superscript II protocol (Invitrogen). Aliquots (2 μl) of the reverse transcription reaction are amplified by PCR. Fragments of the appropriate target SARS gene, i.e., a gene encoding a SARS enzyme, are amplified in 30 cycles (UL54 and UL8: 3 min, 94° C. hot start; 1 min, 94° C. denaturation; 1 min, 55° C. annealing; 1 min, 72° C. polymerization. UL13: 3 min, 94° C. hot start; 1 min, 94° C. denaturation; 1 min, 60° C. annealing; 1 min, 72° C. polymerization) by PCR (Taq-Polymerase, Stratagene), in a 100-μl reaction volume with the appropriate oligonucleotides, as described herein at 0.1 nmol each. 8-μl aliquots of cycle 20-30 (lanes 2-12) of the PCR were resolved on a 2% agarose gel (Invitrogen) according to the manufacturer's instructions.

Cell-based assays of the invention may optionally use a variant or derivative of a wild-type SARS virus that has reduced or attenuated virulence in humans and/or animal models (e.g., mouse, non-human primate, etc.) Use of such attenuated SARS viruses in screening methods may reduce safety concerns and precautions that would otherwise be associated with the pathogenic nature of the SARS virus and may eliminate or reduce the need for the implementation of cumbersome high containment levels during performance of the assays and screening of compounds.

The invention includes an enzyme-based assay that can be used to screen for and identify a therapeutically active agent for the treatment of SARS virus infection.

An embodiment of the invention is an assay comprising contacting a known quantity of SARS protease in solution to a peptide containing a detectable marker and cleavage site for SARS protease, wherein SARS protease activity is monitored by measuring the intensity of the marker on the cleaved product.

In a more particular embodiment, a method of assaying for SARS protease is provided comprising contacting a sample solution containing SARS protease with a peptide containing a fluorescent donor, fluorescent quencher, and cleavage site for SARS protease, said peptide being detectable with a fluorometer when cleaved, wherein SARS protease activity is determined in the sample by the amount of fluorescence detected by the fluorometer.

Assays based on the direct measurement of SARS protease inhibition may be utilized for screening for SARS therapeutics. Protease for such assays such as 3C-like protease and papain-like protease may be isolated and purified for such assays as described in Seybert, et al., J. Gen. Virol., 78:71-75, 1997, Ziebuhr, et al., Adv. Exp. Med. Biol., 440:115-120, 1998, Sims, et al., Adv. Exp. Med. Biol. 440:129-134, 1998, Ziebuhr, et al., J. Virol., 73:177-185, 1999, Teng, et al., J. Virol., 73:2658-2666, 1999, Herold, et al., J. Biol. Chem. 274:14918-14925, 1999, and Ziebuhr, et al., J. Biol. Chem. 276:33220-33232, 2001. Furthermore, Example 30 describes a novel method of purifying SARS protease using column chromatography. Example 31 describes a continuous fluorescence resonance energy transfer (FRET) assay for measuring SARS protease activity. Protease enzyme based assays such as the FRET assay demonstrated in Example 31 are readily adapted for high-throughput screening and are used for screening candidate antiviral compounds. Performance of the protease enzymatic assay in the presence of a SARS protease inhibitor compound will show a decreased amount of fluorescence at a given time when compared to negative control assay containing no test compound on a non-inhibiting control compound. Such a method would involve the steps of: (a) providing an assay solution comprising SARS protease; (b) adding a test compound to the assay solution; (c) adding a substrate for SARS protease to the assay solution; and (d) measuring the proteolytic activity in the assay solution. In a preferred embodiment, the proteolytic activity is measured by the fluorescence of fluorophore product produced by the enzymatic activity of SARS protease.

Attenuated SARS virus variants generally contain one or more genome modifications or mutations (e.g., substitutions, deletions, insertions) in protein encoding or non-coding regions. Specific examples of attenuating mutations include, for example, genetic modifications in the 5′-end noncoding region, leader sequence, intergenic regions, 3′-end noncoding region, ORF 1a, ORF 1b, S gene, E gene, M gene, N gene, or any of the nonstructural protein genes outside of the ORF 1a/1b region. Preferred attenuating mutations are in a SARS virus structural protein (e.g., Spike (S)), a protease or polymerase domain, or a non-coding sequence (e.g., 5′-end noncoding region, intergenic sequence). In addition, a cleavage site may be introduced or eliminated within the spike protein (see for example, Gombold et al., J. Virol. 67:4504-4512, 1993; Bos et al., Virology 214:453-463, 1995), such modification that may also be useful for optimization of expression of recombinant spike protein antigen (e.g., for vaccine purposes).

A variety of methods are used according to the present invention in order to obtain attenuated variants of SARS virus. Such methods include serial passage of the SARS virus in cultured cells (e.g., mammalian cell culture, such as fetal rhesus kidney cells or VERO cells), until the SARS virus demonstrates attenuated function. The serial propagation of virus may be performed at any temperature at which tissue culture passage attenuation occurs, and may be performed in conjunction with one or more steps of mutagenesis (e.g., chemical mutagenesis). The attenuated phenotype of SARS virus variants, obtained after one or more cell culture passages, is readily measured by one skilled in the art. As used herein, attenuation refers to the decreased virulence of the SARS virus in a human subject. Evidence of attenuated function may be indicated by decreased levels of viral replication or by decreased virulence in an animal model.

Other methods of producing an attenuated SARS virus include cell culture passage of the virus at sub-optimal temperatures (cold passage), as well as introduction of attenuating mutations into the SARS viral genome by random mutagenesis (e.g., chemical mutagenesis, such as using 5-fluorouracil) or using directed mutagenesis. Preparation and generation of attenuated RSV vaccines (the methods of which will generally applicable to SARS virus) are disclosed in, for example, EP 0640128, U.S. Pat. No. 6,284,254, U.S. Pat. No. 5,922,326, U.S. Pat. No. 5,882,651.

The number of passages required to obtain safe, immunizing attenuated virus is dependent at least in part on the conditions employed. Periodic testing of the SARS virus culture for virulence and immunizing ability in animals (e.g., mouse, primate) can readily determine the parameters for a particular combination of tissue culture and temperature.

In another embodiment, the cell-based assay for screening of antiviral compounds is based on the readout of expression of a gene product (e.g., reporter gene product) that is not from SARS virus. Gene products particularly suitable to the present invention include, but are not limited to those of the above-described assays.

In order to achieve such a read-out, the gene-of-interest (GOI) encoding said gene reporter gene product must be incorporated into a replicating SARS virus genome or construct derived from a SARS virus genome (e.g., SARS virus replicon, SARS virus defective-interfering (DI) RNA). FIG. 13 is a schematic depicting locations for incorporation of the reporter gene into a SARS virus genome. Preferably, insertion of a heterologous reporter gene-of-interest is at a site between existing SARS virus genes, such as for example, as shown in FIG. 13. For example, the GOI may be inserted closely following the termination codon of a SARS virus gene (e.g., ORF 1b, S, E, M, N). Insertion should be positioned in order to minimize disruption of mRNA transcription for the SARS virus gene(s). The GOI may also be inserted as an in-frame “fusion” with an existing SARS virus gene, such that sufficient function of the GOI is maintained for detection. To optimize expression, an additional SARS virus intergenic sequence (e.g., SEQ ID NO: 7388, with or without additional flanking SARS virus sequences) may also be engineered into a position preceding the inserted GOI.

Incorporation of a GOI into SARS virus may be accomplished by one of skill in the art using a variety of techniques. For example, one preferred method is targeted RNA recombination, that takes advantage of the ability of coronavirus RNAs to undergo recombination within the cell (see for example Fischer et al., J. Virol. 71:5148-5160, 1997; Koljesar et al., J. Vet. Sci. 2:149-157, 2001). A construct of desired configuration (e.g., cDNA of defective interfering RNA of SARS virus) containing the GOI flanked by SARS virus sequence (e.g., intergenic sequence) is generated such that RNA may be transcribed directly within a eukaryotic cell or in vitro and transfected into susceptible cells also infected with SARS virus. Recombinant virus containing the GOI is identified based on expression of the GOI encoded marker.

Alternatively, incorporation of a GOI into SARS virus may be accomplished by one of skill in the art by first assembling a full-length cDNA clone of the SARS virus, that can be used to produce infectious RNA transcripts in vivo (e.g., from an RNA polymerase II promoter) or in vitro (e.g., from a bacteriophage promoter). Although relatively long in genome length, such assembly of a full-length cDNA clone is now readily obtainable by one of skill in the art using standard molecular biology and reverse genetics techniques and the genome sequence of SARS virus (see for example, Thiel et al., J. Gen. Virol., 82:1273-1281, 2001; Almazan et al., Proc. Natl. Acad. Sci. USA 97:5516-5521, 2000; Thiel et al. (2003) J Gen Virol 82:1273-1281; Yount et al (2003) PNAS USA 100:12995-13000). Insertion of a heterologous GOI into a full-length SARS virus genome cDNA may be performed using a variety of techniques, such as for example, ligation into natural or synthetic restriction sites, PCR (e.g., overlapping PCR), and recombination.

It may also be desirable to utilize similar SARS virus recombinants containing a gene-of-interest for antiviral screening, however, with further modification-to minimize or eliminate virus-induced cytopathology (e.g., CPE). Non-cytopathic derivatives from SARS virus may be obtained by one of skill in the art using a variety of methods. For example, a selectable marker (e.g., drug resistance marker) may be incorporated as GOI into a SARS virus genome to produce infectious virus as described above (see for example, Perri et al., J. Virol., 74:9802-9807, 2000). Infectious GOI-containing SARS virus or infectious genome RNA/cDNA is then used to infect/transfect cells (e.g., VERO), with or without prior mutagenesis, after which time the infected cells are subjected to the appropriate selection. Only those cells containing SARS virus harboring both the selectable marker and one or more mutations rendering the virus non-cytopathic will survive the selection process and grow out. Active SARS virus replication in these cells is readily detected using a variety of detection techniques (e.g., PCR, Northern blot) and such cells may serve as the substrate for cell-based screening assays. Mutations that result in the desired noncytopathic SARS virus phenotype may include nucleotide substitutions, deletions or additions, and may occur in a variety of genome coding or non-coding regions (e.g., 5′ or 3′-end noncoding regions, intergenic regions, ORF1a, ORF1b, a protease domain, a polymerase domain). The identification of such mutations is readily accomplished by exchange of sequences with wild-type (e.g., parental) SARS virus and demonstrating transfer of the phenotype, and sequencing of the appropriate genome region. Similar mutations that reduce or eliminate cytopathogenicity also may be utilized in the context of a SARS virus derived replicon vector, either by similar selection directly using a SARS virus replicon or by specific engineering of the replicon based on mutation(s) identified in the context of infectious SARS virus as described above. In addition, such mutations may serve as the basis for attenuated SARS virus derivatives, as described elsewhere in this document.

Alternatively, rather than using infectious SARS virus or its derivatives for cell-based screening assays, propagation defective “replicons” may be engineered and utilized. Such replicons maintain all protein encoding sequences and cis replication sequences required for RNA replication and expression within a cell, but are deleted of one or more sequences or genes required for packaging of progeny SARS virus (see for example Curtis et al., J. Virol., 76:1422-1434, 2002). FIG. 14 is a schematic depicting representative examples of SARS virus replicons according to the present invention. For example a SARS virus cDNA construct is generated, that is lacking one or more (or all) structural protein encoding genes, whereby the missing SARS virus gene(s) is/are replaced by the GOI, maintaining all necessary transcription signals for expression of the GOI. Operably linked with the SARS virus replicon cDNA construct is a promoter for RNA polymerase that can be used to transcribe the replicon RNA in vivo (e.g., RNA polymerase II promoter) or in vitro (e.g., bacteriophage promoter). The SARS replicon may be introduced into a susceptible cell by transfection as RNA or DNA, depending on the promoter of choice, and the transfected cells may be utilized for the evaluation of antiviral compounds. By incorporating one or more mutations rendering the replicon noncytopathic for the cells (see above), one can avoid the need for nucleic acid transfection each time an assay is to be performed.

Alternatively, SARS virus replicons may be packaged into virus like particles that allow infection of cells, rather than requiring transfection of nucleic acid molecules. A requirement for replicon packaging is that essential SARS virus gene functions deleted from the replicon (e.g., one or more structural proteins) are provided in trans within the cell containing the replicon. A variety of methods for packaging of replicon RNA can be utilized to one of skill in the art (see for example, Curtis et al., ibid: Ortego, et al., J. Virol., 76:11518-11529, 2002). For example, stably transformed cell lines constitutively or inducibly expressing the required SARS virus gene functions may be utilized. Alternatively, the required SARS virus gene functions may be expressed by viral vectors that are introduced into the replicon-containing cell. Alternatively a defective interfering (DI) SARS virus derived RNA containing the required gene functions may be introduced into the replicon-containing cell. Such DI constructs used to complement missing replicon functions may be more commonly referred to as defective helper RNA or defective helpers.

Another configuration useful for cell-based antiviral screening assays according to the present invention utilizes SARS virus derived DI RNAs encoding a GOI (see for example Stirrups, et al., J. Gen. Virol., 81:1687-1698, 2000; Liao, et al., Virology 208:319-327, 1995). Introduction of a SARS DI, either as cDNA linked to an RNA polymerase II promoter or as in vitro transcribed RNA, into susceptible cells also infected with SARS virus, allows for a readout of the GOI reporter product in assays.

A replicon-based system for rapid identification of coronavirus replicase inhibitors is described by Hertzig et al. (2004) J Gen Virol DOI 10.1099/vir/0/80044-0. Briefly, the system uses a non-cytopathic selectable replicon RNA that can be stably maintained in eukaryotic cells. The replicon RNA mediates reporter gene expression as a marker for coronavirus replication, and expression of the reporter can be used to test the inhibitory effect of test compounds in vitro, thereby allowing high throughput screening for replicase inhibitors without the need to grow infectious virus. Preferred replicon RNAs include a neomycin resistance gene in the replicase gene with a downstream reporter gene (e.g. GFP) that is expressed via replicase-mediated synthesis of a sub-genomic mRNA.

VI. Compositions and Methods for Treatment of SARS Virus Infection

The present invention relates to compositions and methods for the treatment and/or prevention of SARS. The invention further includes a method for the treatment and/or prevention of SARS through the administration of a therapeutically effective amount of at least one antiviral compound from among those described in the US patents and published international patent applications listed in Table 1 and Table 2. In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In a further embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. Combined treatment with the lopinavir/ritonavir (Kaletra) protease inhibitor and ribavirin has shown a favorable clinical response (Chu et al. (2004) Thorax 59:252-256). In another embodiment, the antiviral compound is an inhibitor of an RNA dependent RNA polymerase. In another embodiment, a first antiviral compound that is a protease inhibitor is administered with a second antiviral compound that is an RNA-dependent RNA polymerase inhibitor. The invention further provides for the administration of a steroidal anti-inflammatory drug in combination with at least one antiviral compound, for example, from the antiviral compounds described in the documents listed in Table 1 and Table 2. A combination treatment of steroids and ribavirin has been described by Fujii et al. (2004) J Infect Chemother 10:1-7. A combination treatment of corticosteroids and interferon alfacon-1 has also been reported (Loutfy et al. (2003) JAMA 290:3222-3228).

The invention further provides for a method for the treatment and/or prevention of SARS through the administration of a therapeutically effective amount of at least one antiviral compound from among those described in the US Patents and published international patent applications listed in Table 1 and Table 2 by inhalation. In another aspect, the antiviral compound may be administered in combination with a SMIP, SMIS, or other immunomodulatory compound such as those in Table 34 and in Table 35. In one embodiment of the method, the antiviral compound is a small molecule. In another embodiment, the antiviral compound is a protease inhibitor. In a further embodiment, the antiviral protease inhibitor is a 3C-like protease inhibitor and/or a papain-like protease inhibitor. In another embodiment, the antiviral compound is an inhibitor of an RNA dependent RNA polymerase. In another embodiment, a first antiviral compound that is a protease inhibitor is administered with a second antiviral compound that is an RNA-dependent RNA polymerase inhibitor. The invention further provides for the administration of a steroidal anti-inflammatory drug in combination with at least one antiviral compound, for example, from the antiviral compounds described in the documents listed in Table 1 and Table 2. The steroidal anti-inflammatory drug may be administered by inhalation for a local effect or administered for systemic absorption such as via an oral or intravenous route.

The invention further provides for methods for treating SARS infection comprising administering a small molecule immunopotentiator (SMIP) compound either alone or in combination with an antiviral compound or in combination with a SARS vaccine. In a further embodiment, the SMIP is a compound disclosed herein or set forth in Table 34.

The invention further provides for methods for treating SARS infection comprising administering an immunosuppressant compound, optionally a small molecule suppressant (SMIS) compound either alone or in combination with an antiviral compound. In a further embodiment, the immunosuppressant compound is disclosed herein or set forth in Table 35.

The invention further provides peptidic immunomodulating compositions, that include oligo and polypeptides, capable of effecting inflammatory response in a patient. In one embodiment, the peptidic immunomodulating composition is able to stimulate human cells to produce cytokines. In another embodiment the peptidic immunomodulating composition is capable of decreasing cytokine levels in the human. Preferred Examples of peptidic immunomodulating compositions include those listed in Table 35, as well as TGFβ2, TGFβ1, TGFβ3, thymopentin (TP5), β-mercaptopropionyl-arginyl-lysyl-aspartyl-valyl-tyrosyl-cysteine amide, colostrinine, lactoferrin (LF), cyclolinopeptide A (CLA), and tuftsin (TKPR). The peptidic immunomodulating compositions of the invention may be used alone or in combination with other agents, preferably antiviral compounds, for the treatment of SARS.

The invention further provides for a kit for use by a consumer for the treatment and/or prevention of SARS. Such a kit comprises: a) a pharmaceutical composition comprising a therapeutically effective amount of at least one antiviral, SMIP, SMIS, or other immunomodulating compound from among those described in the US patents and published international patent applications listed in Table 1, Table 2, Table 34 and Table 35 and a pharmaceutically acceptable carrier, vehicle or diluent; b) a container for holding the pharmaceutical composition; and, optionally, c) instructions describing a method of using the pharmaceutical compositions for the treatment and or the prevention of SARS. The kit may optionally contain a plurality of compounds for the treatment of SARS wherein the antiviral compounds are selected from 3C-like protease inhibitors and papain-like protease inhibitors. In a further embodiment, the kit contains an antiviral compound that is an RNA-dependent RNA polymerase inhibitor. When the kit comprises more than one antiviral, SMIP, SMIS, or other immunomodulating compound, the compounds contained in the kit may be optionally combined in the same pharmaceutical composition.

An additional aspect of the invention provides for the use of at least-one of the antiviral, SMIP, SMIS, or other immunomodulating compounds described in the US Patents and published international patent applications listed in Table 1, Table 2, Table 34 and Table 35 for the manufacture of a medicament for the treatment or prevention of SARS.

An additional aspect of the invention provides for the use of at least one SMIP compound, or at least one immunosuppressant compound, or at least one SMIS compound for the manufacture of a medicament for the treatment or prevention of SARS. Preferred SMIP, immunosuppressant, and SMIS compounds are described herein.

Unless otherwise specified, the following terms, when used within Section VI: “Compositions and Methods for Treatment of SARS Virus Infection” of the present application have the meanings as defined below:

As used herein, “limit”, “treat” and “treatment” are interchangeable terms as are “limiting” and “treating” and, as used herein, include preventative (e.g., prophylactic) and palliative treatment or the act of providing preventative or palliative treatment. The terms include a postponement of development of SARS symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop following infection with a SARS virus. The terms further include ameliorating existing SARS symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms.

Representative uses of the compositions and methods of the present invention include: the elimination or reduction of the viral load of the SARS virus in a vertebrate, including humans, the elimination or reduction of symptoms associated with SARS, and a reduction in morbidity associated with SARS. In a SARS patient population, the use of the compositions and methods of the invention will result in the reduction in the high mortality rates associated with SARS.

Infection with the SARS virus and the symptoms associated with SARS can be treated in a subject by administering the compositions of the invention. The compositions of the invention may be administered systemically. For systemic use, the compounds herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intranasal or transdermal) or enteral (e.g., oral or rectal) delivery according to conventional methods. Intravenous administration can be by a series of injections or by continuous infusion over an extended period. Administration by injection or other routes of discretely spaced administration can be performed at intervals ranging from weekly to once to three times daily or more. Alternatively, the compositions disclosed herein may be administered in a cyclical manner (administration of disclosed composition, followed by no administration, followed by administration of disclosed compositions, and the like). Treatment will continue until the desired outcome is achieved.

A “subject” is a vertebrate animal including a human that is in need of treatment with the compositions, methods and kits of the present invention. The term “subject” or “subjects” is intended to refer to both the male and female gender unless one gender is specifically indicated.

“Coadministration” of a combination of a plurality of antiviral compounds means that these components can be administered together as a composition or as part of the same, unitary dosage form. “Co-administration” also includes administering a plurality of antiviral compounds separately but as part of the same therapeutic treatment program or regimen. “Co-administration” also includes administering a plurality of other agents, such as, for example an oligopeptide, a polypeptide, a peptidic immunomodulator, nucleic acid, antibodies, or a vaccine wherein the compounds or agents are administered separately but as part of the same therapeutic treatment program or regimen. The components need not necessarily be administered at essentially the same time, although they can if so desired. “Co-administration” also includes separate administration at different times and in any order. For example, where appropriate a patient may take one or more component(s) of the treatment in the morning and the one or more of the other component(s) at night.

By “antiviral compound” as used herein is meant an antiviral compound as described in the US patents and published international patent applications listed in Table 1 and Table 2. The US patents and published international patent applications listed in Table 1, Table 2 and Table 35 are incorporated herein in their entirety. In one embodiment, the antiviral compound is an RNA-dependent RNA polymerase. In another preferred embodiment the antiviral compound is a 3C-like protease inhibitor or a papain-like protease inhibitor. The antiviral compounds may be administered in the form of the acid, or of a soluble alkali metal salt or alkaline earth metal salt where appropriate.

The precise dosage of the antiviral compound will vary with the dosing schedule, the oral potency of the particular antiviral compound chosen, the age, size, sex and condition of the subject, the severity of the disorder to be treated, and other relevant medical and physical factors. Thus, a precise pharmaceutically effective amount cannot be specified in advance and can be readily determined by the caregiver or clinician.

Generally, an appropriate amount of antiviral compound is chosen to obtain a reduction in the load of the SARS virus in the subject and/or to obtain a reduction in the symptoms associated with SARS. For humans, an effective oral dose of antiviral compound is typically from about 1.5 to about 6000 μg/kg body weight per day and preferably about 10 to about 2000 μg/kg of body weight per day.

One of ordinary skill in the art will recognize that certain antiviral, SMIP, SMIS, and immunomodulating compounds of the invention including 3C-like protease inhibitors, papain-like protease inhibitators, and RNA-dependent RNA polymerase inhibitors will contain one or more atoms that may be in a particular stereochemical, tautomeric, or geometric configuration, giving rise to stereoisomers, tautomers and configurational isomers. All such isomers and mixtures thereof are included in this invention, when active. Crystalline and amorphous forms of the antiviral compounds of this invention are also included as are hydrates, solvates, polymorphs, and isomorphs of the antiviral compounds of the invention.

SMIP compounds of the invention include compounds are described in issued U.S. Pat. Nos. 4,547,511 and 4,738,971 with the general structure (a):

for the treatment of disorders responsive to agents that enhance cell-mediated immunity.

Immunostimulatory oligonucleotides and polynucleotides are described in PCT WO 98/55495 and PCT WO 98/16247. U.S. Patent Application No. 2002/0164341 describes adjuvants including an unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant. U.S. Patent Application No. 2002/0197269 describes compositions comprising an antigen, an antigenic CpG-ODN and a polycationic polymer.

Additionally, issued U.S. Pat. Nos. 4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, 5,525,612, WO99/29693 and U.S. Ser. No. 09/361,544 disclose compounds of the general structure (b):

for the use as “immune response modifiers.”

Further compounds with SMIP and antiviral activity are described below and in US patent application entitled Thiosemicarbazones as Anti-Virals and Immunopotentiators filed on Dec. 29, 2003 with an attorney docket number of PP19814.004US generally disclosing compounds of the following structures:

A compound of formula c:

wherein: E is absent or selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, and substituted heteroaryl;

L is absent or is selected from the group consisting of oxo, amino, alkylene, substituted alkylene, alkoxy, alkylamino, aminoalkyl, heterocyclyl, carbocyclyl, and carbonyl;

W is absent or selected from the group consisting of cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, and substituted heteroaryl;

X is absent or is selected from the group consisting of oxo, amino, alkylene, substituted alkylene, alkoxy, alkylamino, aminoalkyl, heterocyclyl, carbocyclyl, and carbonyl;

Y is selected from the group consisting of cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, and substituted heteroaryl;

Y′ is absent or is selected from the group consisting of F, Cl, Br, I, nitro, alkyl, substituted alkyl, and optionally substituted heterocyclyl, amino, alkylamino, dialkylamino;

Y″ is absent or is selected from the group consisting of F, Cl, Br, I, nitro, alkyl, substituted alkyl, and optionally substituted heterocyclyl, amino, alkylamino, dialkylamino;

R′ is H, alkyl, or substituted alkyl;

R″ is H, or

R′ and R″ are taken together to form a hetercyclic ring;

Z and Z′ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, alkoxy, substituted alkoxy, aminocarbonyl, alkoxycarbonyl, carboxyl sulfonyl, methanesulfonyl, and substituted or unsubstituted alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, heteroaralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, loweralkylcarbonyl, loweralkoxycarbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, cycloamidino, cycloalkyl, cycloimido, arylsulfonyl and arylsulfonamido; or

Z and Z′ are taken together to form a heterocyclic group, that may be optionally substituted and the tautomers and the pharmaceutically acceptable salts, esters, or prodrugs thereof.

Further SMIP compounds are described below and in U.S. patent application Ser. No. 10/762,873, Use of Tryptanthrin Compounds for Immune Potentiation, filed on Jan. 21, 2004 and disclosing the general embodiment of compounds represented by Formula (d):

wherein

A, B, C, D, E, F, G, and H are independently selected from carbon and nitrogen, or A and B and/or C and D can be taken together to be nitrogen or sulfur;

R₁, R₂, R₃, R₄, R₈, and R₁₀ are independently selected from the group consisting of hydrogen, halogen, loweralkyl, alkyl, substituted alkyl, cycloalkyl, heterocyclyl, alkylheterocyclyl, substituted heterocyclyl, substituted alkenyl, amino, (substituted alkyl)(alkyl)amino, imino, haloloweralkyl, hydroxy, alkoxy, substituted alkoxy, hydroxyalkylthio, nitro, alkylsulfonyl, N-alkylsulfonamide, arylalkyl, arylalkylaryl, arylaryl, aryloxy, arylamino, acylamino, acyloxyamino, alkylaminoacylamino, alkylaminosulfonylamino, alkylamino, alkenylamino, dialkylamino, alkoxyalkylamino, alkoxyalkylheterocyclyl, mercaptoalkoxyalkyl, cyano, formyl, —COOR₁₁ wherein R₁₁ is hydrogen, loweralkyl, aryl, heterocyclyl, monosaccharide or disaccharide, and —CONR₁₂R₁₃ wherein R₁₂ and R₁₃ are independently selected from hydrogen, loweralkyl, aryl, heterocyclyl, saccharide, peptide and amino acid residues; or R₂ and R₃ taken together form a six membered aromatic ring;

R₇ and R₉ are independently selected from hydrogen, halogen, loweralkyl, haloloweralkyl, cycloalkyl, heterocyclyl, substituted heterocyclyl or heterocyclylalkyl; and

R₁, R₂, R₃, R₄, R₇, R₈, R₉, and R₁₀ are absent when the ring atom to which they would otherwise be bonded is sulfur or double-bonded nitrogen; or

the a pharmaceutically acceptable salts, esters, or prodrugs thereof, provided that R₁, R₂, R₃, R₄, R₇, R₈, R₉, and R₁₀ are not all hydrogen when A, B, C, D, E, F, and H are carbon.

In one embodiment, the compounds of Formula (I) have a backbone structure wherein D is nitrogen, and A-C and E-H are carbon.

In one embodiment, when D is carbon, at least one, or at least two of R₁—R₄, and R₇—R₁₀ are not hydrogen.

In one embodiment, R₁ through R₄, and R₈ and R₁₀ are independently selected from at least two of the group consisting of hydrogen, halogen, loweralkyl, cycloalkyl, heterocyclyl, substituted heterocyclyl, alkylheterocyclyl, amino, imino, haloloweralkyl, alkoxy, nitro, alkylsulfonyl, arylalkyl, arylalkylaryl, arylaryl, aryloxy, arylamino, acylamino, acyloxyamino, alkylaminoacylamino, alkylaminosulfonylamino, alkylamino, alkenylamino, dialkylamino, alkoxyalkylamino, alkoxyalkylheterocyclyl, mercaptoalkoxyalkyl, cyano, formyl, —COOR₁₁ where R₁₁ is hydrogen, loweralkyl, aryl, heterocyclyl, monosaccharide or disaccharide, and —CONR₁₂R₁₃ where R₁₂ and R₁₃ are independently selected from hydrogen, loweralkyl, aryl, heterocyclyl, saccharide, peptide and amino acid residues; and R₄ is not present when D is nitrogen.

In an additional embodiment, 4A, B, C, D, E, F, G, and H are independently selected from carbon and nitrogen;

R₁, R₂, R₃, R₄, R₈ and R₁₀ are independently selected from the group consisting of hydrogen, halogen, loweralkyl, alkyl, substituted alkyl, heterocyclyl, substituted heterocyclyl, substituted alkenyl, (substituted alkyl)(alkyl)amino, haloloweralkyl, hydroxy, alkoxy, substituted alkoxy, hydroxyalkylthio, nitro, N-alkylsulfonamide, cyano, —COOR₁₁ wherein R₁₁ is hydrogen, loweralkyl, aryl, heterocyclyl, monosaccharide or disaccharide, and —CONR₁₂R₁₃ wherein R₁₂ and R₁₃ are independently selected from hydrogen, loweralkyl, aryl, heterocyclyl, saccharide, peptide and amino acid residues.

For the compounds described herein:

The term “loweralkyl” refers to branched or straight chain acyclical alkyl groups comprising one to ten carbon atoms, including, e.g., methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and the like.

The term “alkyl” refers to alkyl groups that do not contain heteroatoms. Thus the term includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following that are provided by way of example: —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃, —C(CH₂CH₃)₃, —CH₂CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂, —CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂, —CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃, —CH₂CH₂C(CH₂CH₃), —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)₂, —CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. The term also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted with straight and branched chain alkyl groups as defined above. The term also includes polycyclic alkyl groups such as, but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl and such rings substituted with straight and branched chain alkyl groups as defined above. Thus, the phrase unsubstituted alkyl groups includes primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. Unsubstituted alkyl groups may be bonded to one or more carbon atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the parent compound. Preferred unsubstituted alkyl groups include straight and branched chain alkyl groups and cyclic alkyl groups having 1 to 20 carbon atoms. More preferred such unsubstituted alkyl groups have from 1 to 10 carbon atoms while even more preferred such groups have from 1 to 5 carbon atoms. Most preferred unsubstituted alkyl groups include straight and branched chain alkyl groups having from 1 to 3 carbon atoms and include methyl, ethyl, propyl, and —CH(CH₃)₂.

The phrase “substituted alkyl” refers to an unsubstituted alkyl group as defined above in which one or more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen and non-carbon atoms such as, but not limited to, a halogen atom in halides such as F, Cl, Br, and I; a phosphorus atom in groups such as phosphate and dialkyl alkylphosphonate; oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as in trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. Substituted alkyl groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom is replaced by a bond to a heteroatom such as oxygen in carbonyl, carboxyl, and ester groups; nitrogen in groups such as imines, oximes, hydrazones, and nitriles. Preferred substituted alkyl groups include, among others, alkyl groups in which one or more bonds to a carbon or hydrogen atom is/are replaced by one or more bonds to fluorine atoms. One example of a substituted alkyl group is the trifluoromethyl group and other alkyl groups that contain the trifluoromethyl group. Other alkyl groups include those in which one or more bonds to a carbon or hydrogen atom is replaced by a bond to an oxygen atom such that the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxy group. Still other alkyl groups include alkyl groups that have an amine, alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine,diarylamine, heterocyclylamine, (alkyl)(heterocyclyl)amine, (aryl)(heterocyclyl)amine, or diheterocyclylamine group.

The term “alkoxy” refers to RO— wherein R, for example, is alkyl such as loweralkyl defined above. Representative examples of loweralkyl alkoxy groups include methoxy, ethoxy, t-butoxy and the like.

The phrase “substituted alkoxy” refers to RO—, where R is, for example, an alkyl substituted, for example, with a halogen. RO is for example OCF₃.

The term “alkenyl” refers to a branched or straight chain groups comprising two to twenty carbon atoms that also comprises one or more carbon-carbon double bonds. Representative alkenyl groups include prenyl, 2-propenyl (i.e., allyl), 3-methyl-2-butenyl, 3,7-dimethyl-2,6-octadienyl, 4,8-dimethyl-3,7-nonadienyl, 3,7,11-trimethyl-2,6,10-dodecatrienyl and the like.

The phrase “substituted alkenyl” refers to alkenyl groups that are substituted, for example, diethyl hex-5-enylphosponate, and others with an alkyl or substituted alkyl group such as dialkyl phosphate or an ester such as an acetate ester.

The phrase “dialkyl amino” refers to an amino group substituted with two alkyl groups such as C1-20 alkyl groups.

The phrase “substituted dialkyl amino” refers to a dialkylamino substituted, for example, with a carboxylic acid, ester, hydroxy or alkoxy.

The term “hydroxyalkylthio” refers to a thio radical to which is appended a hydroxyalkyl group, where the alkyl is for example lower alkyl. An example is hydroxyethylthio, —SCH₂CH₂OH.

The term “N-alkylsulfonamide” refers to the group —SO₂NHalkyl, where alkyl is, for example, octyl.

The term “alkynyl” refers to a branched or straight chain comprising two to twenty carbon atoms that also comprises one or more carbon-carbon triple bonds. Representative alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.

The term “aryl” refers to aryl groups that do not contain heteroatoms. Thus the term includes, but is not limited to, groups such as phenyl, biphenyl, anthracenyl, naphthenyl by way of example. Although the phrase “unsubstituted aryl” includes groups containing condensed rings such as naphthalene, it does not include aryl groups that have other groups such as alkyl or halo groups bonded to one of the ring members, as aryl groups such as tolyl are considered herein to be substituted aryl groups as described below. A preferred unsubstituted aryl group is phenyl. Unsubstituted aryl groups may be bonded to one or more carbon atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the parent compound, however.

The phrase “substituted aryl group” has the same meaning with respect to aryl groups that substituted alkyl groups had with respect to alkyl groups. However, a substituted aryl group also includes aryl groups in which one of the aromatic carbons is bonded to one of the non-carbon or non-hydrogen atoms described above and also includes aryl groups in which one or more aromatic carbons of the aryl group is bonded to a substituted and/or unsubstituted alkyl, alkenyl, or alkynyl group as defined herein. This includes bonding arrangements in which two carbon atoms of an aryl group are bonded to two atoms of an alkyl, alkenyl, or alkynyl group to define a fused ring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus, the phrase “substituted aryl” includes, but is not limited to tolyl, and hydroxyphenyl among others.

The term “arylalkyl” refers to a loweralkyl radical to which is appended an aryl group. Representative arylalkyl groups include benzyl, phenylethyl, hydroxybenzyl, fluorobenzyl, fluorophenylethyl and the like.

The phrase “unfused arylaryl” refers to a group or substituent to which two aryl groups, that are not condensed to each other, are bound. Exemplary unfused arylaryl compounds include, for example, phenylbenzene, diphenyldiazene, 4-methylthio-1-phenylbenzene, phenoxybenzene, (2-phenylethynyl)benzene, diphenyl ketone, (4-phenylbuta-1,3-diynyl)benzene, phenylbenzylamine, (phenylmethoxy)benzene, and the like. Preferred substituted unfused arylaryl groups include: 2-(phenylamino)-N-[4-(2-phenylethynyl)phenyl]acetamide, 1,4-diphenylbenzene, N-[4-(2-phenylethynyl)phenyl]-2-[benzylamino]acetamide, 2-amino-N-[4-(2-phenylethynyl)phenyl]propanamide, 2-amino-N-[4-(2-phenylethynyl)phenyl]acetamide, 2-(cyclopropylamino)-N-[4-(2-phenylethynyl)phenyl]acetamide, 2-(ethylamino)-N-[4-(2-phenylethynyl)phenyl]acetamide, 2-[(2-methylpropyl)amino]-N-[4-(2-phenylethynyl)phenyl]acetamide, 5-phenyl-2H-benzo[d]1,3-dioxolene, 2-chloro-1-methoxy-4-phenylbenzene, 2-[(imidazolylmethyl)amino]-N-[4-(2-phenylethynyl)phenyl]acetamide, 4-phenyl-1-phenoxybenzene, N-(2-aminoethyl)[4-(2-phenylethynyl)phenyl]carboxamide, 2-{[(4-fluorophenyl)methyl]amino}-N-[4-(2-phenylethynyl)phenyl]acetamide, 2-{[(4-methylphenyl)methyl]amino}-N-[4-(2-phenylethynyl)phenyl]acetamide, 4-phenyl-1-(trifluoromethyl)benzene, 1-butyl-4-phenylbenzene, 2-(cyclohexylamino)-N-[4-(2-phenylethynyl)phenyl]acetamide, 2-(ethylmethylamino)-N-[4-(2-phenylethynyl)phenyl]acetamide, 2-(butylamino)-N-[4-(2-phenylethynyl)phenyl]acetamide, N-[4-(2-phenylethynyl)phenyl]-2-(4-pyridylamino)acetamide, N-[4-(2-phenylethynyl)phenyl]-2-(quinuclidin-3-ylamino)acetamide, N-[4-(2-phenylethynyl)phenyl]pyrrolidin-2-ylcarboxamide, 2-amino-3-methyl-N-[4-(2-phenylethynyl)phenyl]butanamide, 4-(4-phenylbuta-1,3-diynyl)phenylamine, 2-(dimethylamino)-N-[4-(4-phenylbuta-1,3-diynyl)phenyl]acetamide, 2-(ethylamino)-N-[4-(4-phenylbuta-1,3-diynyl)phenyl]acetamide, 4-ethyl-1-phenylbenzene, 1-[4-(2-phenylethynyl)phenyl]ethan-1-one, N-(1-carbamoyl-2-hydroxypropyl)[4-(4-phenylbuta-1,3-diynyl)phenyl]carboxamide, N-[4-(2-phenylethynyl)phenyl]propanamide, 4-methoxyphenyl phenyl ketone, phenyl-N-benzamide, (tert-butoxy)-N-[(4-phenylphenyl)methyl]carboxamide, 2-(3-phenylphenoxy)ethanehydroxamic acid, 3-phenylphenyl propanoate, 1-(4-ethoxyphenyl)-4-methoxybenzene, and [4-(2-phenylethynyl)phenyl]pyrrole.

The phrase “unfused heteroarylaryl” refers to a unfused arylaryl group where one of the aryl groups is a heteroaryl group. Exemplary heteroarylaryl groups include, for example, 2-phenylpyridine, phenylpyrrole, 3-(2-phenylethynyl)pyridine, phenylpyrazole, 5-(2-phenylethynyl)-1,3-dihydropyrimidine-2,4-dione, 4-phenyl-1,2,3-thiadiazole, 2-(2-phenylethynyl)pyrazine, 2-phenylthiophene, phenylimidazole, 3-(2-piperazinylphenyl)furan, 3-(2,4-dichlorophenyl)-4-methylpyrrole, and the like. Preferred substituted unfused heteroarylaryl groups include: 5-(2-phenylethynyl)pyrimidine-2-ylamine, 1-methoxy-4-(2-thienyl)benzene, 1-methoxy-3-(2-thienyl)benzene, 5-methyl-2-phenylpyridine, 5-methyl-3-phenylisoxazole, 2-[3-(trifluoromethyl)phenyl]furan, 3-fluoro-5-(2-furyl)-2-methoxy-1-prop-2-enylbenzene, (hydroxyimino)(5-phenyl(2-thienyl))methane, 5-[(4-methylpiperazinyl)methyl]-2-phenylthiophene, 2-(4-ethylphenyl)thiophene, 4-methylthio-1-(2-thienyl)benzene, 2-(3-nitrophenyl)thiophene, (tert-butoxy)-N-[(5-phenyl(3-pyridyl))methyl]carboxamide, hydroxy-N-[(5-phenyl(3-pyridyl))methyl]amide, 2-(phenylmethylthio)pyridine, and benzylimidazole.

The phrase “unfused heteroarylheteroaryl” refers to an unfused arylaryl group where both of the aryl groups is a heteroaryl group. Exemplary heteroarylheteroaryl groups include, for example, 3-pyridylimidazole, 2-imidazolylpyrazine, and the like. Preferred substituted unfused heteroarylheteroaryl groups include: 2-(4-piperazinyl-3-pyridyl)furan, diethyl(3-pyrazin-2-yl(4-pyridyl))amine, and dimethyl {2-[2-(5-methylpyrazin-2-yl)ethynyl](4-pyridyl)}amine.

The phrase “fused arylaryl” refers to an aryl group as previously defined that is condensed, and fully conjugated to an aryl group. Representative fused arylaryl groups include biphenyl, 4-(1-naphthyl)phenyl, 4-(2-naphthyl)phenyl and the like.

The phrase “fused heteroarylaryl” refers to an aryl group as previously defined that is condensed, and fully conjugated with a heteroaryl group. Representative fused heteroarylaryl groups include quinoline, quinazoline and the like.

The phrase “fused heteroarylheteroaryl” refers to a heteroaryl group as previously defined that is condensed, and fully conjugated with another heteroaryl group. Representative fused heteroarylheteroaryl groups include pyrazalopyrimidine, imidazoquinoline and the like.

The term “aryloxy” refers to RO— wherein R is an aryl group. Representative arylalkoxy group include benzyloxy, phenylethoxy and the like.

The term “arylalkoxy” refers to a lower alkoxy radical to which is appended an aryl group. Representative arylalkoxy group include benzyloxy, phenylethoxy and the like.

The term “aryloxyaryl” refers to an aryl radical to which is appended an aryloxy group. Representative aryloxyaryl groups include 4-phenoxyphenyl, 3-phenoxyphenyl, 4-phenoxy-1-naphthyl, 3-phenoxy-1-naphthyl and the like.

The term “aryloxyarylalkyl” refers to an arylalkyl radical to which is appended an aryloxy group. Representative aryloxyarylalkyl groups include 4-phenoxyphenylmethyl, 3-phenoxyphenylmethyl, 4-phenoxyphenylethyl, 3-phenoxy-phenylethyl and the like.

The term “arylalkoxyaryl” refers to an aryl radical to which is appended an arylalkoxy group. Representative arylalkoxyaryl groups include 4-benzyloxylphenyl, 3-benzyloxyphenyl and the like.

The term “arylalkoxyarylalkyl” refers to an arylalkyl radical to which is appended an arylalkoxy group. Representative arylalkoxyarylalkyl groups include 4-benzyloxylbenzyl, 3-benzyloxybenzyl and the like.

The term “cycloalkyl” refers to an alicyclic group comprising from 3 to 7 carbon atoms including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “cycloalkylalkyl” refers to a loweralkyl radical to which is appended a cycloalkyl group. Representative examples of cycloalkylalkyl include cyclopropylmethyl, cyclohexylmethyl, 2-(cyclopropyl)ethyl and the like.

The term “halogen” refers to iodine, bromine, chlorine or fluorine; “halo” refers to iodo, bromo, chloro or fluoro.

The term “haloalkyl” refers to a lower alkyl radical, as defined above, bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like.

The term “heterocyclyl” (or heterocyclic, or heterocyclo) refers to both aromatic and nonaromatic ring compounds including monocyclic, bicyclic, and polycyclic ring compounds such as, but not limited to, quinuclidyl, containing 3 or more ring members of which one or more is a heteroatom such as, but not limited to, N, O, and S. Although the phrase “unsubstituted heterocyclyl” includes condensed heterocyclic rings such as benzimidazolyl, it does not include heterocyclyl groups that have other groups such as alkyl or halo groups bonded to one of the ring members as compounds such as 2-methylbenzimidazolyl are substituted heterocyclyl groups. Examples of heterocyclyl groups include, but are not limited to: unsaturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms such as, but not limited to pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g. 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl etc.), tetrazolyl, (e.g. 1H-tetrazolyl, 2H tetrazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms such as, but not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensed unsaturated heterocyclic groups containing 1 to 4 nitrogen atoms such as, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl; unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl (e.g. 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to, morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl, benzoxadiazolyl, benzoxazinyl (e.g. 2H-1,4-benzoxazinyl etc.); unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1 to 3 nitrogen atoms-such as, but not limited to, thiazolyl, isothiazolyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, thiazolodinyl; saturated and unsaturated 3 to 8 membered rings containing 1 to 2 sulfur atoms such as, but not limited to, thienyl, dihydrodithiinyl, dihydrodithionyl, tetrahydrothiophene, tetrahydrothiopyran; unsaturated condensed heterocyclic rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g. 2H-1,4-benzothiazinyl, etc.), dihydrobenzothiazinyl (e.g. 2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated 3 to 8 membered rings containing oxygen atoms such as, but not limited to furyl; unsaturated condensed heterocyclic rings containing 1 to 2 oxygen atoms such as benzodioxolyl (e.g. 1,3-benzodioxoyl, etc.); unsaturated 3 to 8 membered rings containing an oxygen atom and 1 to 2 sulfur atoms such as, but not limited to, dihydrooxathiinyl; saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as 1,4-oxathiane; unsaturated condensed rings containing 1 to 2 sulfur atoms such as benzothienyl, benzodithiinyl; and unsaturated condensed heterocyclic rings containing an oxygen atom and 1 to 2 oxygen atoms such as benzoxathiinyl. Heterocyclyl group also include those described above in which one or more S atoms in the ring is double-bonded to one or two oxygen atoms (sulfoxides and sulfones). For example, heterocyclyl groups include tetrahydrothiophene, tetrahydrothiophene oxide, and tetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5 or 6 ring members. More preferred heterocyclyl groups include morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine, thiomorpholine in which the S atom of the thiomorpholine is bonded to one or more O atoms, pyrrole, homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, and tetrahydrofuran.

The phrase “substituted heterocyclyl” refers to an heterocyclyl group as defined above in which one of the ring members is bonded to a non-hydrogen atom such as described above with respect to substituted alkyl groups and substituted aryl groups. Examples, include, but are not limited to, 2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl, 1-methyl piperazinyl, and 2-chloropyridyl among others.

“Aminosulfonyl” refers to the group -S(O) 2-NH2. “Substituted aminosulfonyl” refersto the group —S(O)₂—NRR′ where R is loweralkyl and R′ is hydrogen or a loweralkyl. The term “aralkylaminosulfonlyaryl” refers to the group -aryl-S(O)₂—NH-aralkyl, where the aralkyl is loweraralkyl.

“Carbonyl” refers to the divalent group —C(O)—.

“Carbonyloxy” refers generally to the group —C(O)—O—,. Such groups include esters, —C(O)—O—R, where R is loweralkyl, cycloalkyl, aryl, or loweraralkyl. The term “carbonyloxycycloalkyl” refers generally to both an “carbonyloxycarbocycloalkyl” and an “carbonyloxyheterocycloalkyl”, i.e., where R is a carbocycloalkyl or heterocycloalkyl, respectively. The term “arylcarbonyloxy” refers to the group —C(O)—O-aryl, where aryl is a mono- or polycyclic, carbocycloaryl or heterocycloaryl. The term “aralkylcarbonyloxy” refers to the group —C(O)—O-aralkyl, where the aralkyl is loweraralkyl.

The term “sulfonyl” refers to the group —SO₂—. “Alkylsulfonyl” refers to a substituted sulfonyl of the structure —SO₂R— in which R is alkyl. Alkylsulfonyl groups employed in compounds of the present invention are typically loweralkylsulfonyl groups having from 1 to 6 carbon atoms in its backbone structure. Thus, typical alkylsulfonyl groups employed in compounds of the present invention include, for example, methylsulfonyl (i.e., where R is methyl), ethylsulfonyl (i.e., where R is ethyl), propylsulfonyl (i.e., where R is propyl), and the like. The term “arylsulfonyl” refersto the group —SO₂-aryl. The term “aralkylsulfonyl” refers to the group —SO₂-aralkyl, in which the aralkyl is loweraralkyl. The term “sulfonamido” refers to —SO₂NH₂.

The term “carbonylamino” refers to the divalent group —NH—C(O)— in which the hydrogen atom of the amide nitrogen of the carbonylamino group can be replaced a loweralkyl, aryl, or loweraralkyl group. Such groups include moieties such as carbamate esters (—NH—C(O)—O—R) and amides —NH—C(O)—O—R, where R is a straight or branched chain loweralkyl, cycloalkyl, or aryl or loweraralkyl. The term “loweralkylcarbonylamino” refers to alkylcarbonylamino where R is a loweralkyl having from 1 to about 6 carbon atoms in its backbone structure. The term “arylcarbonylamino” refers to group —NH—C(O)—R where R is an aryl. Similarly, the term “aralkylcarbonylamino ” refers to carbonylamino where R is a lower aralkyl.

The term “guanidino” or “guanidyl” refers to moieties derived from guanidine, H2N—C(═NH)—NH₂. Such moieties include those bonded at the nitrogen atom carrying the formal double bond (the “2”-position of the guanidine, e.g., diaminomethyleneamino, (H2N)₂C═NH—) and those bonded at either of the nitrogen atoms carrying a formal single bond (the “1-” and/or “3”-positions of the guandine, e.g., H₂N—C(═NH)—NH—). The hydrogen atoms at any of the nitrogens can be replaced with a suitable substituent, such as loweralkyl, aryl, or loweraralkyl.

Representative cycloimido and heterocycloimido groups include, for example, those shown below. These cycloimido and heterocycloimido can be further substituted and may be attached at various positions as will be apparent to those having skill in the organic and medicinal chemistry arts in conjunction with the disclosure herein.

Representative substituted amidino and heterocycloamidino groups include, for example, those shown below. These amidino and heterocycloamidino groups can be further substituted as will be apparent to those having skill in the organic and medicinal chemistry arts in conjunction with the disclosure herein.

Representative substituted alkylcarbonylamino, alkyloxycarbonylamino, aminoalkyloxycarbonylamino, and arylcarbonylamino groups include, for example, those shown below. These groups can be further substituted as will be apparent to those having skill in the organic and medicinal chemistry arts in conjunction with the disclosure herein.

Representative substituted aminocarbonyl groups include, for example, those shown below. These can heterocyclo groups be further substituted as will be apparent to those having skill in the organic and medicinal chemistry arts in conjunction with the disclosure herein.

Representative substituted alkoxycarbonyl groups include, for example, those shown below. These alkoxycarbonyl groups can be further substituted as will be apparent to those having skill in the organic and medicinal chemistry arts in conjunction with the disclosure herein.

“Substituted” refers to the definite replacement of hydrogen with one or more monovalent or divalent radicals. Suitable substitution groups include, those described herein for particular groups, as well as hydroxyl, nitro, amino, imino, cyano, halo, thio, thioamido, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, alkyl, substituted alkyl, haloloweralkyl, loweralkoxy, haloloweralkoxy, loweralkoxyalkyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio, aminoalkyl, cyanoalkyl, benzyl, pyridyl, pyrazolyl, pyrrole, thiophene, imidazolyl, and the like.

The term “linking moiety” refers to a covalent bond or an uncyclized divalent group, such as, for example, —CO—, —O—, —S—, —CH₂—, —NH—, and substituted or unsubstituted alkyl, alkenyl, alkynyl, carbonyl, alkoxycarbonyl groups as defined herein.

The term “SMIP compound” refers to small molecule immunopotentiating compounds, that include small molecule compounds below about MW 1000 g/mol, preferably MW 800 g/mol that are capable of stimulating or modulating a pro-inflammatory response in a patient. In an embodiment, the SMIP compounds are able to stimulate human peripheral blood mononuclear cells to produce cytokines. Preferred SMIP compounds and derivatives thereof include, for example, aminoazavinyl compounds, benzazole compounds, acylpiperazine compounds, indoledione compounds, tetrahydroisoquinoline (THIQ) compounds, anthraquinone compounds, indanedione compounds, pthalimide compounds, benzocyclodione compounds, aminobenzimidazole quinolinone (ABIQ) compounds, hydraphthalimide compounds, pyrazolopyrimidine compounds, quinazilinone compounds, quinoxaline compounds, triazine compounds, tetrahydropyrrolidinoquinoxaline compounds, pyrrole compounds, benzophenone compounds, sterol compound, and isoxazole compounds.

The term “SMIS compound” refers to small molecule immunosuppressant compounds, that include small molecule compounds below about about MW 1000 g/mol, preferably MW 800 g/mol, capable of suppressing or modulating a pro-inflammatory response in a patient.

Acylpiperazine compounds as described throughout this application include compounds of formula (III) as shown below:

wherein,

-   -   R₉ is selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, arylalkyl, arylalkenyl,         heteroarylalkyl, and heteroarylalkenyl;     -   R₁₀ is substituted or unsubstituted alkyl;     -   n is an integer from 0-2; and     -   if D₁ is carbon than D₂ is oxygen, D₃ is absent, and D₄ is         selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, carbocycyl, alkoxyaryl, fused         arylaryl, fused arylheteroaryl, and fused heteroarylaryl; or,     -   if D₁ is nitrogen than D₂ is nitrogen, D₄ is absent, and D₃ is         selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, carbocycyl, alkoxyaryl, fused         arylaryl, fused arylheteroaryl, and fused heteroarylaryl.

Indoledione compounds as described throughout this application include compounds of formula (IV) as shown below:

wherein,

-   -   R₁₁ and R₁₂ are independently selected from the group consisting         of H, nitro, halogen, amino, hydroxy, cyano, carboxcyclic acid,         and substituted or unsubstituted alkyl, aryl, heteroaryl,         alkoxy, alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl groups; and,     -   R₁₃ is selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, arylalkyl, heteroarylalkyl,         heterocyclyl, heterocyclylalkyl, and alkylbenzyl.

Tetrahydroisoquinoline (THIQ) compounds as described throughout this application include compounds of formula (V) as shown below:

wherein,

-   -   L is a covalent bond or selected from the group consisting of         —CH₂—, —CO—, —O—, —S—, CHF, —NH—, —NR₂₀—, where R₂₀ is lower         alkyl;     -   R₁₄ is selected from the group consisting of hydrogen, halogen,         and substituted or unsubstituted alkyl;     -   R₁₅ is selected from the group consisting of substituted or         unsubstituted carbocyclyl, aryl, arylalkyl, alkoxyaryl,         heteroaryl, heterocyclyl;     -   R₁₆ is selected from the group consisting of hydrogen, halogen,         and substituted or unsubstituted alkyl;     -   R₁₇ is selected from the group consisting of hydrogen, halogen,         and substituted or unsubstituted alkyl; and,     -   P₁₈ and R₁₉ are independently selected from the group consisting         of H, hydroxy, halogen, alkoxy, amino, unsubstituted alkyl,         substituted alkyl, and alkylamino.

Benzocyclodione compounds as described throughout this application include compounds of formula (VI) as shown below:

wherein,

-   -   E is selected from the group consisting of NR₂₅ or CR₂₆R₂₇;     -   R₂₁, R₂₃, and R₂₄ are independently selected from the group         consisting of H, hydroxy, halogen, alkoxy, amino, unsubstituted         alkyl, substituted alkyl, and alkylamino;     -   R₂₂ is selected from the group consisting or H, hydroxy,         halogen, alkoxy, amino, and unsubstituted or substituted alkyl,         and alkylamino, arylalkyl, heteroarylalkyl, aryl, heteroaryl,         arylcarbonyl, heterocyclyl, heterocyclylalkyl, and         heteroarylcarbonyl;     -   R₂₅ is selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, heterocyclyl, carbocyclyl,         arylalkyl, heteroarylalkyl, and heterocyclyalkyl;     -   R₂₆ is selected from the group consisting of H, halogen,         hydroxy, amino, and substituted or unsubstituted alkyl,         carbonylalkyl, and alkylcarbonylalkyl; and,     -   R₂₇ is selected from the group aryl, arylalkyl, heteroarylalkyl,         heterocyclyl, heterocyclylalkyl, carbocyclyl, arylcarbonylalkyl,         and arylalkylcarbonyl.

Aminoazavinyl compounds as described throughout this application include compounds of formula (VII) as shown below:

wherein,

-   -   G is either S or NH;     -   R₂₈ is selected from the group consisting of H, and substituted         or unsubstituted alkyl, aryl, heteroaryl, heteroarylalkyl,         arylalkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, and         heterocyclylalkyl;     -   Q is selected from the group consisting of hydrogen, substituted         alkyl, unsubstituted alkyl, and aryl, substituted aryl,         heteroaryl, substituted heteroaryl, heterocyclyl, substituted         heterocyclyl, fused or unfused arylaryl, substituted arylaryl,         arylheteroaryl, substituted arylheteroaryl,         heteroarylheteroaryl, and substituted heteroarylheteroaryl;     -   V₁ is selected from the group consisting of alkyl, substituted         alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,         heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted         heteroarylalkyl, alkoxy, substituted alkoxy, aminocarbonyl,         alkoxycarbonyl, carboxyl sulfonyl, methanesulfonyl, and         substituted or unsubstituted alkylcarbonyl, arylcarbonyl,         aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl,         alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy,         heteroarylcarbonyloxy, heteroaralkylcarbonyloxy,         alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl,         loweralkylcarbonyl, loweralkoxycarbonyl, aminocarbonyl,         aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino,         heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino,         arylaminocarbonylamino, aralkylcarbonylamino,         heteroarylcarbonylamino, arylcarbonylamino, cycloamidino,         cycloalkyl, cycloimido, arylsulfonyl and arylsulfonamido; and,     -   V₂ is selected from the group consisting of hydrodgen, halogen,         alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,         substituted arylalkyl, heteroaryl, substituted heteroaryl,         heteroarylalkyl, substituted heteroarylalkyl, alkoxy,         substituted alkoxy, aminocarbonyl, alkoxycarbonyl, carboxyl         sulfonyl, methanesulfonyl, and substituted or unsubstituted         alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,         heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy,         arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy,         heteroaralkylcarbonyloxy, alkylaminocarbonyloxy,         arylaminocarbonyloxy, formyl, loweralkylcarbonyl,         loweralkoxycarbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl,         sulfonamido, aminoalkoxy, alkylamino, heteroarylamino,         alkylcarbonylamino, alkylaminocarbonylamino,         arylaminocarbonylamino, aralkylcarbonylamino,         heteroarylcarbonylamino, arylcarbonylaamino, cycloamidino,         cycloalkyl, cycloimido, arylsulfonyl and arylsulfonamido.

Lactam compounds as described throughout this application include compounds of formula (VIII) as shown below:

wherein,

-   -   W₁ is selected from the group consisting of —OH, —OR₃₆ groups,         —NR₃₇R₃₈;     -   W₂ is selected from the group consisting of O, S, and NR₃₉         groups;     -   R₂₉ and R₃₀ join to form a 5 to 6 membered substituted or         unsubstituted ring comprising all carbon atoms or at least one         O, N, or S atom;     -   R₃₅ and R₃₉ may be the same or different and are selected from         the group consisting of H, —OH substituted and unsubstituted         alkyl groups, substituted and unsubstituted aryl groups,         —C(═O)H, —C(═O)-alkyl groups, and —C(═O)-aryl groups;     -   R₃₁, R₃₂, R₃₃, and R₃₄ may be the same or different and are         independently selected from the group consisting of H, Cl, Br,         F, I, —NO₂, —CN, —OH, —OR₄₀ groups, —NR₄₁R₄₂ groups, —C(═O)R₄₃         groups, —SH groups, substituted and unsubstituted amidinyl         groups, substituted and unsubstituted guanidinyl groups,         substituted and unsubstituted alkyl groups, substituted and         unsubstituted aryl groups, substituted and unsubstituted alkenyl         groups, substituted and unsubstituted alkynyl groups,         substituted and unsubstituted heterocyclyl groups, substituted         and unsubstituted alkylaminoalkyl groups, substituted and         unsubstituted dialkylaminoalkyl groups, substituted and         unsubstituted arylaminoalkyl groups, substituted and         unsubstituted diarylaminoalkyl groups, substituted and         unsubstituted (alkyl)(aryl)aminoalkyl groups, substituted and         unsubstituted heterocyclylalkyl groups, substituted and         unsubstituted aminoalkyl groups, substituted and unsubstituted         heterocyclylaminoalkyl groups, substituted and unsubstituted         diheterocyclylaminoalkyl groups, substituted and unsubstituted         (alkyl)(heterocyclyl)aminoalkyl groups, substituted and         unsubstituted (aryl)(heterocyclyl)aminoalkyl groups, substituted         and unsubstituted hydroxyalkyl groups, substituted and         unsubstituted alkoxyalkyl groups, substituted and unsubstituted         aryloxyalkyl groups, and substituted and unsubstituted         heterocyclyloxyalkyl groups;     -   R₃₆ is selected from the group consisting of substituted and         unsubstituted alkyl groups, substituted and unsubstituted aryl         groups, substituted and unsubstituted heterocyclyl groups,         substituted and unsubstituted heterocyclylalkyl groups, —C(═O)H,         —C(═O)-alkyl groups, —C(═O)-aryl groups, —C(═O)O-alkyl groups,         —C(═O)O-aryl groups, —C(═O)NH₂, —C(═O)NH(alkyl) groups,         —C(═O)NH(aryl) groups, —C(═O)N(alkyl)₂ groups, —C(═O)N(aryl)₂         groups, —C(═O)N(alkyl)(aryl) groups, —NH₂, —NH(alkyl) groups,         —NH(aryl) groups, —N(alkyl)₂ groups, —N(alkyl)(aryl) groups,         —N(aryl)₂ groups, —C(═O)NH(heterocyclyl) groups,         —C(═O)N(heterocyclyl)₂ groups, —C(═O)N(alkyl)(heterocyclyl)         groups, and —C(═O)N(aryl)(heterocyclyl) groups;     -   R₃₇ is selected from the group consisting of H, substituted and         unsubstituted alkyl groups, substituted and unsubstituted aryl         groups, and substituted and unsubstituted heterocyclyl groups;     -   R₃₈ is selected from the group consisting of H, substituted and         unsubstituted alkyl groups, substituted and unsubstituted aryl         groups, substituted and unsubstituted heterocyclyl groups, —OH,         alkoxy groups, aryloxy groups, —NH₂, substituted and         unsubstituted heterocyclylalkyl groups, substituted and         unsubstituted aminoalkyl groups, substituted and unsubstituted         alkylaminoalkyl groups, substituted and unsubstituted         dialkylaminoalkyl groups, substituted and unsubstituted         arylaminoalkyl groups, substituted and unsubstituted         diarylaminoalkyl groups, substituted and unsubstituted         (alkyl)(aryl)aminoalkyl groups, substituted and unsubstituted         alkylamino groups, substituted and unsubstituted arylamino         groups, substituted and unsubstituted dialkylamino groups,         substituted and unsubstituted diarylamino groups, substituted         and unsubstituted (alkyl)(aryl)amino groups, —C(═O)H,         —C(═O)-alkyl groups, —C(═O)-aryl groups, —C(═O)O-alkyl groups,         —C(═O)O-aryl groups, —C(═O)NH₂, —C(═O)NH(alkyl) groups,         —C(═O)NH(aryl) groups, —C(═O)N(alkyl)₂ groups, —C(═O)N(aryl)₂         groups, —C(═O)N(alkyl)(aryl) groups, —C(═O)-heterocyclyl groups,         —C(═O)—O-heterocyclyl groups, —C(═O)NH(heterocyclyl) groups,         —C(═O)-N(heterocyclyl)₂ groups, —C(═O)-N(alkyl)(heterocyclyl)         groups, —C(═O)-N(aryl)(heterocyclyl) groups, substituted and         unsubstituted heterocyclylaminoalkyl groups, substituted and         unsubstituted diheterocyclylaminoalkyl groups, substituted and         unsubstituted (alkyl)(heterocyclyl)aminoalkyl groups,         substituted and unsubstituted (aryl)(heterocyclyl)aminoalkyl         groups, substituted and unsubstituted hydroxyalkyl groups,         substituted and unsubstituted alkoxyalkyl groups, substituted         and unsubstituted aryloxyalkyl groups, and substituted and         unsubstituted heterocyclyloxyalkyl groups;     -   R₄₁ is selected from the group consisting of H, substituted and         unsubstituted alkyl groups, substituted and unsubstituted aryl         groups, and substituted and unsubstituted heterocyclyl groups;     -   R₄₂ is selected from the group consisting of H, substituted and         unsubstituted alkyl groups, substituted and unsubstituted aryl         groups, substituted and unsubstituted heterocyclyl groups,         —C(═O)H, —C(═O)-alkyl groups, —C(═O)-aryl groups, —C(═O)NH₂,         —C(═O)NH(alkyl) groups, —C(═O)NH(aryl) groups, —C(═O)N(alkyl)₂         groups, —C(═O)N(aryl)₂ groups, —C(═O)N(alkyl)(aryl) groups,         —C(═O)O-alkyl groups, —C(═O)O-aryl groups, substituted and         unsubstituted aminoalkyl groups, substituted and unsubstituted         alkylaminoalkyl groups, substituted and unsubstituted         dialkylaminoalkyl groups, substituted and unsubstituted         arylaminoalkyl groups, substituted and unsubstituted         diarylaminoalkyl groups, substituted and unsubstituted         (alkyl)(aryl)aminoalkyl groups, substituted and unsubstituted         heterocyclylalkyl groups, —C(═O)-heterocyclyl groups,         —C(═O)—O-heterocyclyl groups, —C(═O)NH(heterocyclyl) groups,         —C(═O)—N(heterocyclyl)₂ groups, —C(═O)—N(alkyl)(heterocyclyl)         groups, —C(═O)—N(aryl)(heterocyclyl) groups, substituted and         unsubstituted heterocyclylaminoalkyl groups, substituted and         unsubstituted diheterocyclylaminoalkyl groups, substituted and         unsubstituted (heterocyclyl)(alkyl)aminoalkyl groups,         substituted and unsubstituted (heterocyclyl)(aryl)aminoalkyl         groups, substituted and unsubstituted hydroxyalkyl groups,         substituted and unsubstituted alkoxyalkyl groups, substituted         and unsubstituted aryloxyalkyl groups, and substituted and         unsubstituted heterocyclyloxyalkyl groups; and     -   R₄₃ is selected from the group consisting of H, —NH₂, —NH(alkyl)         groups, —NH(aryl) groups, —N(alkyl)₂ groups, —N(aryl)₂ groups,         —N(alkyl)(aryl) groups, —NH(heterocyclyl) groups,         —N(heterocyclyl)(alkyl) groups, —N(heterocyclyl)(aryl) groups,         —N(heterocyclyl)₂ groups, substituted and unsubstituted alkyl         groups, substituted and unsubstituted aryl groups, —OH,         substituted and unsubstituted alkoxy groups, substituted and         unsubstituted heterocyclyl groups, substituted and unsubstituted         aryloxy groups, heterocyclyloxy groups, —NHOH, —N(alkyl)OH         groups, —N(aryl)OH groups, —N(alkyl)O-alkyl groups,         —N(aryl)O-alkyl groups, —N(alkyl)O-aryl groups, and         —N(aryl)O-aryl groups.

Preferably R₂₉ and R₃₀ join together to form a substituted or unsubstituted phenyl ring.

Hydropthalamide compounds as described throughout this application include compounds of formula (IX) as shown below:

wherein,

-   -   R₄₄ is selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, arylalkyl, heteroarylalkyl,         fused arylaryl, unfused arylaryl, fused heteroarylaryl, unfused         heteroarylaryl, fused arylheteroaryl, and unfused         arylheteroaryl;     -   R₄₅, R₄₇, R₄₉, and R₅₁ may be the same or different and are         independently selected from the group consisting of H, nitro,         halogen, amino, hydroxy, cyano, carboxcyclic acid, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl; and     -   R₄₆, R₄₈, R₅₀, and R₅₂ may be the same or different and are         independently selected from the group consisting of H, halogen,         and substituted or unsubstituted alkyl groups.

Benzophenone compounds as described throughout this application include compounds of formula (X) as shown below:

wherein,

-   -   R₅₃ is independently selected from the group consisting of H,         nitro, halogen, amino, hydroxy, cyano, carboxcyclic acid, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl;     -   R₅₄ is independently selected from the group consisting of H,         nitro, halogen, amino, hydroxy, cyano, carboxcyclic acid, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl; and     -   o and p are integers from 0-4.

Isoxazole compounds as described throughout this application include compounds of formula (XI) as shown below:

wherein,

-   -   R₅₅ is selected from the group consisting of substituted or         unsubstituted aryl, arylalkyl, heteroaryl, heteroarylalkyl,         heterocyclyl, and heterocyclylalkyl;     -   R₅₆ is selected from the group consisting of substituted or         unsubstituted aryl, arylalkyl, heteroaryl, heteroarylalkyl,         heterocyclyl, and heterocyclylalkyl; and,     -   R₅₇ is selected from the group consisting of H, halogen, hydoxy,         and substituted or unsubstituted alkyl, aryl, heteroaryl,         heterocyclyl, and carbonyl.

Sterol compounds as described throughout this application include compounds of formula (XII) as shown below:

wherein,

-   -   R₅₈ is selected from the group consisting of substituted or         unsubstituted aryl, arylalkyl, heteroaryl, heteroarylalkyl,         heterocyclyl, and heterocyclylalkyl.

Preferably R₅₈ is a pyranone substituent.

Quinazilinone compounds as described throughout this application include compounds of formula (XIII) as shown below:

wherein,

-   -   R₅₉ is selected from the group consisting of H, halogen,         hydroxy, and substituted or unsubstituted alkyl, aminoalkyl,         alklyaminoalkyl, alkoxy, dialkylaminoalkyl, hydroxyalkyl,         alkenyl, alkynyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,         and heterocyclylalkyl;     -   R₆₀ is selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, arylalkyl, heteroarylalkyl, and         heterocyclylalkyl; and,     -   R₆₁, R₆₂, R₆₃, and R₆₄ may be the same or different and are         independently selected from the group consisting of H, nitro,         halogen, amino, hydroxy, cyano, carboxcyclic acid, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl groups.

Pyrrole compounds as described throughout this application include compounds of formula (XIV) as shown below:

wherein,

-   -   R₆₅ is selected from the group consisting of H, hydroxy, and         substituted or unsubstituted alkyl, aryl, heteroaryl,         heteroarylalkyl, arylalkyl, heteroarylaminoalkyl,         arylaminoalkyl, heteroaryloxyalkyl, and aryloxyalkyl groups;     -   R₆₆, R₆₇, R₆₈, and R₆₉ may be the same or different and are         independently selected from the group consisting of H, nitro,         halogen, amino, hydroxy, cyano, carboxcyclic acid, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl groups.

Further preferred pyrrole compounds include those shown in Formula (XV):

wherein:

-   -   K₁ is nitrogen, oxygen, or optionally substituted carbon;     -   W is absent or is selected from the group consisting of —O—,         —S—, —S(O)—, —SO₂—, —NH—, —NH—CO—, —NR′CO—, —NHSO₂—, —NR′SO₂—,         —CO—, —CO₂—, —CH₂—, —CF₂—, CHF, —CONH—, —CONR′—, and —NR′—,         where R′ is alkyl, substituted alkyl, cycloalkyl, aryl,         heteroaryl, heterocyclo; Ar is optionally substituted aryl,         heteroaryl, or a protecting group;     -   R₇₀ and R₇₀′ are independently selected from the group         consisting of hydrogen and methyl;     -   R₇₁, R₇₂, R₇₃, and R₇₄ are independently selected from the group         consisting of hydrogen, hydroxyl, and optionally substituted         loweralkyl, cycloloweralkyl, cyclicaminoalkyl, alkylaminoalkyl,         loweralkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl,         aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl         and heteroaryl;     -   R₇₅ and R₇₈ are independently selected from the group consisting         of hydrogen, halo, and optionally substituted loweralkyl,         cycloalkyl, alkoxy, amino, aminoalkoxy, carbonyloxy,         aminocarbonyloxy, alkylcarbonylamino, arylcarbonylamino,         aralkylcarbonylamino, heteroarylcarbonylamino,         heteroaralkylcarbonylamino, cycloimido, heterocycloimido,         amidino, cycloamidino, heterocycloamidino, guanidinyl, aryl,         heteroaryl, heterocycloalkyl, heterocyclocarbonyloxy,         heteroarylcarbonyloxy, and arylsulfonamido;     -   R₇₆ is selected from the group consisting of hydrogen, aryl,         heteroaryl, substituted heteroaryl, heterocyclyl, and         substituted heterocyclyl;     -   R₇₇ is selected from the group consisting of hydrogen, hydroxy,         halo, carboxyl, nitro, amino, amido, amidino, imido, cyano,         sulfonyl, methanesulonyl, and substituted or unsubstituted         alkyl, alkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,         heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy,         arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy,         heteroaralkylcarbonyloxy, alkylaminocarbonyloxy,         arylaminocarbonyloxy, formyl, loweralkylcarbonyl,         loweralkoxycarbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl,         sulfonamido, aminoalkoxy, alkylamino, heteroarylamino,         alkylcarbonylamino, alkylaminocarbonylamino,         arylaminocarbonylamino, aralkylcarbonylamino,         heteroarylcarbonylamino, arylcarbonylamino,         heteroarylcarbonylamino cycloamido, cyclothioamido,         cycloamidino, heterocycloamidino, cycloalkyl, cycloimido,         heterocycloimido, guanidinyl, aryl, heteroaryl, heterocyclo,         heterocycloalkyl, arylsulfonyl and arylsulfonamido;

Anthraquinone compounds of the instant invention include, for example, compounds of Formula (XVI):

wherein,

-   -   R₇₉, R₈₀, R₈₁, and R₈₂ may be the same or different and are         independently selected from the group consisting of H, nitro,         halogen, amino, hydroxy, cyano, carboxcyclic acid, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, sulfonyl, aminosulfonyl,         alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy,         alkylamino, arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl groups; and,     -   R₈₃ and R₈₄ are taken together to form a substituted or         unsubstituted 5-6 membered ring containing all carbon atoms or         1-2 heteroatoms selected from the group consisting of O, S, and         N.

Quinoxaline compounds referred to throughout this application include tricyclic, partially unconjugated compounds optionally substituted with nitrogen heteroatoms as shown in the preferred quinoxaline embodiment (XVII) below:

wherein,

-   -   J₁ is either C or N,     -   J₁′ is selected from the group consisting of H, substituted         aryl, unsubstituted aryl, substituted heteroaryl, and         unsubstituted heteroaryl;     -   J₂ is either C or N,     -   J₂′ is selected from the group consisting of H, substituted         aryl, unsubstituted aryl, substituted heteroaryl, and         unsubstituted heteroaryl;     -   J₃ is selected from the group consisting of —CO—, —NH—, and —N═;     -   if J₄ is —O— then J₄′ is absent; or,     -   if J₄ is ═C— then J₄′ is selected from the group consisting of H         and substituted or unsubstituted alkyl, alkoxy, aryl,         heteroaryl, heteroarylalkyl, arylalkyl, aminoalkyl, alkylamino,         and alkylthio groups; and,     -   R₈₅, R₈₆, R₈₇, R₈₈, and R₈₉ may be the same or different and are         independently selected from the group consisting of H, nitro,         halogen, amino, hydroxy, cyano, carboxcyclic acid, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, sulfonyl, aminosulfonyl,         alkylaminocarbonyl, aminocarbonyl, arylalkoxy, heteroarylalkoxy,         alkylamino, arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl groups.

Triazine compounds refer to substituted 6-membered heterocyclic groups with 3 nitrogen atoms distributed throughout the ring. The preferred embodiments of the instant invention include those shown in structures (XVIII), (XIX) and (XX) shown below:

wherein,

-   -   R₉₀ is selected from the group consisting of substituted or         unsubstituted alkyl, alkenyl, akynyl, aryl, heteroaryl,         heteroarylalkyl, heteroarylalkenyl, arylalkyl, and arylalkenyl;     -   R₉₁ and R₉₃ are independently selected from the group consisting         of H, and unsubstituted alkyl;     -   R₉₁ is aryl; preferably phenyl,         wherein,     -   R₉₄ is selected from the group consisting of H, amino, alkyl,         aminoalkyl, and halogen;     -   R₉₅ is selected from the group consisting of substituted or         unsubstituted aryl, arylamino, arylalkylamino, heteroaryl,         heteroarylamino, and heteroalkylamino;     -   R₉₆ and R₉₇ are independently selected from the group consisting         of H, halogen, and alkyl, preferably methyl; or,     -   R₉₆ may form a double bond with the nitrogen atom directly below         it as indicated by the dashed line in the above structure; and,         wherein,

R₉₈ is selected from the group consisting of H, substituted alkyl, and unsubstituted alkyl; preferably methyl,

-   -   R₉₉ is selected from the group consisting of H, substituted         alkyl, and unsubstituted alkyl; preferably ethyl,     -   R₁₀₀ is selected from the group consisting of substituted or         unsubstituted aryl, heteroaryl, alkoxyaryl, arylalkyl, and         heteroarylalkyl.

Benzazole compounds as described throughout this application include compounds of formula (XXI) as shown below:

wherein,

-   -   A is selected from the group consisting of —O—, —S—, —NH—, and         —NR₈—;     -   W is selected from the group consisting of —CH₂—, —O—, —S—,         —NH—, and —NR₈—;     -   R₇ is selected from the group consisting of carbocyclyl, unfused         carbocyclylcarbocyclyl, substituted aryl, unsubstituted aryl,         substituted heteroaryl, unsubstituted heteroaryl, substituted         fused arylheteroaryl, unsubstituted fused arylheteroaryl,         substituted unfused arylaryl and unsubstituted unfused arylaryl;     -   R₆ is selected from the group consisting of substituted or         unsubstituted aryl, and heteroaryl; and,     -   R₈ is independently substituted or unsubstituted alkyl.

Pyrazalopyrimidine compounds as described throughout this application include compounds of formula (XXII) as shown below:

wherein,

-   -   R₁₀₁ is selected from the group consisting of H, nitro, halogen,         amino, hydroxy, cyano, carboxcyclic acid, and substituted or         unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl,         alkylcarbonylamino, sulfonyl, aminosulfonyl, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl groups;     -   R₁₀₂ is selected from the group consisting of H, nitro, halogen,         amino, hydroxy, cyano, carboxcyclic acid, and substituted or         unsubstituted alkyl, aryl, heteroaryl, alkoxy, alkylcarbonyl,         alkylcarbonylamino, alkylaminocarbonyl, aminocarbonyl,         arylalkoxy, heteroarylalkoxy, alkylamino, arylalkylamino,         arylamino, heteroarylamino, heteroarylaminoalkyl, heterocyclyl,         heterocyclylalkoxy, heterocyclylalkyl, and carbocyclyl groups;     -   R₁₀₃ is selected from the group consisting of H, nitro, halogen,         amino, hydroxy, cyano, carboxcyclic acid, trifluoromethyl, and         substituted or unsubstituted alkyl, aryl, heteroaryl, alkoxy,         alkylcarbonyl, alkylcarbonylamino, alkylaminocarbonyl,         aminocarbonyl, arylalkoxy, heteroarylalkoxy, alkylamino,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, and carbocyclyl groups;     -   R₁₀₄ is selected from the group consisting of H and substituted         or unsubstituted aryl, heteroaryl, arylalkoxy, heteroarylalkoxy,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, carbocyclylalkyl and carbocyclyl groups;     -   R₁₀₅ is selected from the group consisting of H and substituted         or unsubstituted aryl, heteroaryl, arylalkoxy, heteroarylalkoxy,         arylalkylamino, arylamino, heteroarylamino,         heteroarylaminoalkyl, heterocyclyl, heterocyclylalkoxy,         heterocyclylalkyl, carbocyclylalkyl and carbocyclyl groups;     -   wherein at least one of R₁₀₄ and R₁₀₅ is not H.

SMIP compounds identified by in-vitro (cellular or non-cellular assays) or in-vivo methods are thoroughly described in Methods 1 and 2 below.

Pharmaceutical compositions containing the compounds of the invention may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers contemplated for use in the practice of the present invention include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, and the like, as well as mixtures of two or more thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, stabilizers, and the like. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like. For parenteral administration, the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like. Compositions of the present invention may also be in the form of microparticles, microcapsules, liposomal encapsulates, and the like, as well as combinations of any two or more thereof.

Other additives include immunostimulatory agents known in the art. Immunostimulatory oligonucleotides and polynucleotides are described in PCT WO 98/55495 and PCT WO 98/16247. U.S. Patent Application No. 2002/0164341 describes adjuvants including an unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant. U.S. Patent Application No. 2002/0197269 describes compositions comprising an antigen, an antigenic CpG-ODN and a polycationic polymer. Other immunostimulatory additives described in the art may be used, for example, as described in U.S. Pat. No. 5,026,546; U.S. Pat. No. 4,806,352; and U.S. Pat. No. 5,026,543.

A controlled release delivery system may be used, such as a diffusion controlled matrix system or an erodible system, as described for example in: Lee, “Diffusion-Controlled Matrix Systems”, pp. 155-198 and Ron and Langer, “Erodible Systems”, pp. 199-224, in “Treatise on Controlled Drug Delivery”, A. Kydonieus Ed., Marcel Dekker, Inc., New York 1992. The matrix may be, for example, a biodegradable material that can degrade spontaneously in situ and in vivo for, example, by hydrolysis or enzymatic cleavage, e.g., by proteases. The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, for example in the form of a hydrogel. Exemplary polymers with cleavable linkages include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imidocarbonates) and poly(phosphazenes).

The compounds of the invention may be administered enterally, orally, parenterally, sublingually, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. For example, suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophoretic, intravenous, intramuscular, intraperitoneal, intranasal, subdermal, rectal, and the like. Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices. The term parenteral includes subcutaneous injections, intravenous, intramuscular, intrastemal injection, or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-propanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

As to the mode of administration, it should be emphasized that it is the combination of therapeutic agents that gives rise to its synergistic therapeutic effect no matter whether the first and the second agent are administered together or separately. Therefore, the two agents may be given together in a single dose or in separate ones with respect to space and time.

Effective amounts of the compounds of the invention generally include any amount sufficient to detectably treat viral infections.

Successful treatment of a subject in accordance with the invention may result in the inducement of a reduction or alleviation of symptoms in a subject afflicted with a medical or biological disorder to, for example, halt the further progression of the disorder, or the prevention of the disorder.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.W., p. 33 et seq (1976).

While the SMIP compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents used in the treatment of SARSs. Other representative agents useful in combination with the compounds of the invention for the treatment of viral infections include, for example, interferon, ribavirin, gancyclovir and the like.

When additional active agents are used in combination with the compounds of the present invention, the additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53^(rd) Edition (1999), that is incorporated herein by reference, or such therapeutically useful amounts as would be known to one of ordinary skill in the art.

The compounds of the invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions of the invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

Compounds of the present invention can be readily synthesized using the methods described herein, or other methods, that are well known in the art.

The compounds can be used in the form of salts derived from inorganic or organic acids. These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-napthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or dispersible products are thereby obtained.

Examples of acids that may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. Basic addition salts can be prepared in situ during the final isolation and purification of the compounds of formula (I), or separately by reacting carboxylic acid moieties with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutical acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine. Pharmaceutical acceptable salts include, but are not limited to, cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

Various compounds and methods of their synthesis are disclosed in international patent application Publication Nos. WO02/18327 (benzamide and pyridylamide based compounds); WO0222598, and WO02/18383 (ABIQ based compounds); and WO 02/81443 (pthalamide base compounds), that have been found within context of this invention to be useful for immune potentiation. The entire disclosure of these U.S. and international publications is incorporated herein by this reference. Other compounds or intermediates of interest in the present invention were purchased from commercially available sources using the following method: the chemical structure of interest was drawn into the ACD-SC database (from MDL Information Systems). A search of the following companies/institutions, among others, retrieved the identified compound's supplier and purchasing information: ASDI, ASINEX, BIONET, CHEMBRIDGE, CHEMDIV, CHEMEX, CHEMSTAR, COMGENEX, CSC, INTERBIOSCREEN, LABOTEST, MAYBRIDGE, MICROSOURCE/GENESIS, OLIVIA, ORION, PEAKDALE, RYAN SCIENTIFIC, SPECS, TIMTEC, U OF FLORIDA, and ZELINSKY.

Benzazle Compounds

Scheme 1

Compounds of the invention containing a benzimidazole core may be prepared using a number of methods familiar to one of skill in the art. In one method, suitably functionalized diamines may be coupled with various thioisocyanates to form the intermediate thioureas. Cyclization to form the benzimidazole moiety may be effected under known conditions such as with treatment carbodiimides or alkyl halides. Alternatively the diamines may be reacted sequentially with carbonyl diimidazole and phosphoryl chloride followed by coupling with the appropriate amine.

Compounds containing the oxazole structure may similarly be prepared according to the methods above or according to other known general procedures. Haviv et. al. (J. Med. Chem. 1988, 31, 1719) describes a procedure for assembling oxazole cores wherein a hydroxy aniline is treated with ethyl potassium xanthate. The resulting sulfuryl benzoxazole may then be chlorinated and coupled with an amine.

Compounds containing a benzothiazole core may also be prepared according to known methods. An ortho-halothioisocyanate may be reacted with an amine to form a thiourea. Reduction with NaH then allows formation of the thiazole ring.

Benzothiazoles may generally be substituted in accordance with the present invention, such as through the following synthetic pathway:

Synthesis of 4-[(2-{[4-chloro-3-(trifluoromethyl)phenyl]amino}-1H-benzimidazol-6-yl)oxyl-N-methylpyridine-2-carboxamide

The compound 4-[(2-{[4-chloro-3-(trifluoromethyl)phenyl]amino}-1H-benzimidazol-6-yl)oxy]-N-methylpyridine-2-carboxamide (159322) was synthesized as follows:

Step 1. Synthesis of 4-[(4-amino-3-nitrophenyl)oxy]-N-methylpyridine-2-carboxamide: A mixture containing 4-amino-3-nitrophenol (1 eq) and potassium bis(trimethylsilyl)amide (2 eq) was stirred in dimethylformamide for 2 hours at room temperature. To this mixture was added (4-chloro(2-pyridyl))-N-methylcarboxamide (1 eq) and potassium carbonate (1.2eq) and stirred at 90° C. for 3 days. The reaction mixture was then concentrated and partitioned between ethyl acetate and water. The organic layer was separated and washed with brine, dried, filtered, and concentrated in vacuum to give brown solid. Purification on silica gel (2% triethyl amine/50% ethyl acetate in hexane) gave 4-[(4-amino-3-nitrophenyl)oxy]-N-methylpyridine-2-carboxamide as an orange solid. The product gave satisfactory NMR. HPLC, 3.39 min; MS: MH⁺=289.

Step 2. Synthesis of 4-[(3,4-diaminophenyl)oxy]-N-methylpyridine-2-carboxamide: The mixture containing [4-(3-amino-4-nitrophenoxy)(2-pyridyl)]-N- in methanol with catalytic amount of 10% Pd/C was hydrogenated until disappearance of the yellow color to yield the product amine. HPLC, 2.5 mins; MS: MH⁺=259.

Step 3. Synthesis of 4-[(2-{[4-chloro-3-(trifluoromethyl)phenyl]amino}-1H-benzimidazol-6-yl)oxy]-N-methylpyridine-2-carboxamide: The mixture containing 4-[(3,4-diaminophenyl)oxy]-N-methylpyridine-2-carboxamide (1 eq) and 4-chloro-3-(trifluoromethyl)benzeneisothiocyanate (1 eq) in tetrahydrofuran was stirred at room temperature for 16 hours to give the corresponding thiourea. To the resulting mixture was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2 eq) and the mixture was stirred for another 10 hours. The mixture was concentrated and partitioned between ethyl acetate and water. The organic layer was washed with brine and dried. Purification on HPLC gave 4-[(2-{[4-chloro-3-(trifluoromethyl)phenyl]amino}-1H-benzimidazol-6-yl)oxy]-N-methylpyridine-2-carboxamide. MS: MH⁺=462

Synthesis of 4-({2-[(4-bromophenyl)amino]-1-methyl-1H-benzimidazol-5-yl}oxy)-N-methylpyridine-2-carboxamide

The compound 4-({2-[(4-bromophenyl)amino]-1-methyl-1H-benzimidazol-5-yl}oxy)-N-methylpyridine-2-carboxamide (161651) was synthesized as follows:

Step 1. Synthesis of 4-{[3-amino-4-(methylamino)phenyl]oxy}-N-methylpyridine-2-carboxamide: A solution of 4-[(4-amino-3-nitrophenyl)oxy]-N-methylpyridine-2-carboxamide (1 eq) in methylene chloride was treated with trifluoroacetic anhydride (I eq) and stirred for 10 minutes at 0° C. The mixture was quenched with satd. NaHCO₃ solution. The organic layer was separated and washed with water, brine, dried and evaporated. MS: MH⁺=385.2

To a solution of the trifluroacetamide (1 eq) in a mixture of toluene, acetonitrile and sodium hydroxide solution (50%) was added benzyltrimethylammonium chloride (1 eq) and dimethyl sulfate (1.2 eq). The biphasic mixture was stirred overnight at room temperature and evaporated. The mixture was taken up in ethyl acetate, washed with water, brine, dried and evaporated. The crude product was purified by column chromatography eluting with 1:1 hexanes and ethylacetate followed by 2% triethylamine in 1:1 hexanes and ethyl acetate followed by 2% triethylamine in 1:1 hexanes and ethyl acetate to afford N-methyl-4-{[4-(methylamino)-3-nitrophenyl]oxy}pyridine-2-carboxamide as a reddish orange solid. MS: MH⁺=303.1.

The solution of nitromethylaniline in methanol was treated with 5% palladium on carbon and stirred under hydrogen atmosphere for 15 min. (until the disappearance of yellow coloration) at room temperature. The mixture was filtered and the filtrate was concentrated to provide 0.36 g of the diamine 4-{[3-amino-4-(methylamino)phenyl]oxy}-N-methylpyridine-2-carboxamide. MS: MH⁺=273.3.

Step 2. Synthesis of 4-({2-[(4-bromophenyl)amino]-1-methyl-1H-benzimidazol-5-yl}oxy)-N-methylpyridine-2-carboxamide: A solution of the diamine 4-{[3-amino-4-(methylamino)phenyl]oxy}-N-methylpyridine-2-carboxamide (1 eq) in methanol was treated with 4-bromophenylisothiocyanate (1 eq) and stirred at 60° C.-65° C. for 2 hours. The reaction mixture was cooled down to room temperature and methyl iodide (1 eq) was added and stirred overnight at 60° C. The reaction was cooled to room temperature, evaporated, taken up in ethyl acetate, and washed with water and brine, dried, and evaporated under reduced pressure. Column chromatography using a gradient solvent system of hexanes and ethyl acetate and either 1:1 methylene chloride and acetone or 5% methanol in methylene chloride yielded the product as a half white powder. MS: MH⁺=452.3

Aminobenzimidazolylquinolinones

Compounds of structure I may be synthesized from simple starting molecules as shown in Schemes 1-4 and exemplified in the Examples. As shown in Scheme 1, compounds of structure I may generally be prepared using aromatic compounds substituted with amines and carboxylic acid groups.

As shown in Scheme 2, a substituted aromatic compound such as a substituted or unsubstituted 2-aminobenzoic acid may be reacted with an acyl halide such as methyl 2-(chlorocarbonyl)acetate to produce an amide that will react with a substituted or unsubstituted 1,2-diaminobenzene. The resulting product is a 4-hydroxy-substituted compound of structure I. One skilled in the art will recognize that the procedure set forth in Scheme 1 may be modified to produce various compounds.

A method for preparing 4-amino substituted compounds of structure I is shown in Scheme 3. As shown in Scheme 3, aromatic compounds substituted with amine and nitrile 20 groups may be used to synthesize 4-amino substituted compounds of structure I. A compound such as ethyl 2-cyanoacetate may be reacted with ethanol to produce ethyl 3-ethoxy-3-iminopropanoate hydrochloride. Subsequent reaction with a substituted or unsubstituted 1,2-phenylenediamine provides substituted or unsubstituted ethyl 2-benzimidazol-2-ylacetate. Reaction of a substituted or unsubstituted ethyl 2-benzimidazol-2-ylacetate with an aromatic compound having an amine and nitrile group such as substituted or unsubstituted 2-aminobenzonitrile with a base such as lithium bis(trimethylsilyl)amide or a Lewis acid such as tin tetrachloride provides the substituted or unsubstituted 4-amino substituted compound of structure I.

Scheme 4 illustrates a general synthetic route that allows for the synthesis of 4-dialkylamino and 4-alkylamino compounds of structure I. An inspection of Scheme 3 shows that 4-hydroxy substituted compounds of structure I may be converted into the 4-chloro derivative by reaction with phosphorous oxychloride or thionyl chloride. The 4-chloro derivative may then be reacted with an alkylamine or dialkylamine to produce the corresponding 4-alkylamino or 4-dialkylamino derivative. Deprotection affords the final 4-alkylamino or 4-dialkylamino compounds of structure I. Other groups that may be reacted with the 4-chloro derivative in this manner include, but are not limited to, ROH, RSH, and CuCN.

As shown in Scheme 5, the synthesis of compounds of structure I having a H, alkyl group, aryl group, or heterocyclyl group in the 4-position may be accomplished using a substituted or unsubstituted 2-benzimidazol-2-ylacetate prepared as shown in Schemes 3 and 4.

Thiosemcarbazones

General procedure for the preparation of thiosemicarbazones

A solution of aldehyde (1.0 equiv.) and thiosemicarbazide (1.05 equiv.) in acetic acid was stirred overnight. Excess of acetic acid was removed to give a residue, that was washed with ethanol, or purified by preparative-HPLC to give the thiosemicarbazone.

Scheme 7

A solution of aldehyde (1.0 equiv.), thiosemicarbazide (1.05 equiv.) and acetic acid (0.1 equiv.) in methanol was stirred overnight. Methanol was removed to give a residue, that was worked up as in Scheme 6.

Scheme 8

To a solution of {[(1E)-1-aza-2-(4-fluoro-3-nitrophenyl)vinyl]aamino}-aminomethane-1-thione in ethanol was added an arylamine (2.1 equiv.). The solution was stirred at room temperature until the starting fluoride disappeared. The solution was purified to the product.

Scheme 9

A mixture of 4-(diethylamino)-2-hydroxybenzaldehyde (1 equiv.), benzylic bromide (1.2 equiv.) and powder potassium carbonate in ethanol was stirred at room temperature for 2 days. Ethanol was removed, and the residue was dissolved in ethyl acetate and water. The organic layer was washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄., and concentrated. The residue was purified on silica gel eluting with ethyl acetate/hexane to give 4-(diethylamino)-2-benzoxylic-benzaldehyde.

The aldehydes were converted to thiosemicarbazones according to Scheme 7.

Scheme 10

A solution of 3,4-difluorobenzenecarbonitrile (1 equiv.), amine (1.5 equiv.) and DIEA (2 equiv.) in NMP was heated in a Smith Microwave (Personal Chemistry) for 30 minutes. The reaction mixture was purified on silica gel to give 4-substituted 3-fluorobenzenecarbonitrile.

To a solution of nitrile in toluene at −78° C. was added DIBAL-H (1 M in toluene, 1.5 equiv.). The reaction mixture was warmed to rt, and stirred for 16 h, and quenched with methanol/ethyl acetate/brine (1:1:4). After being stirred at rt for 30 min, the solution was extracted with ethyl acetate (3×). The combined organic layers were washed with aqueous NaHCO₃, brine and concentrated. The aldehyde was purified on silica gel or directly converted to thiosemicarbazones (Scheme 7).

Scheme 11

A solution of 2,4,5-trifluorobenzenecarbonitrile (1 equiv.) and 4-arylpiperazine (1.2 equiv.) and DIEA (1.2 equiv.) in THF was heated at 80° C. for 2 hours. The mixture was purified on silica gel to give 4-substituted 2,5-difluorobenzenecarbonitrile.

Scheme 12

To an alcohol (1.0 equiv) was added potassium t-butoxide in THF (1 M, 1.1 equiv). After 5 minutes, the solution was added to a solution of 4-N-substituted-2,5-difluorobenzenecarbonitrile (1 equiv.) in THF. The reaction mixture was stirred at rt overnight and quenched with aqueous ammonium chloride. The aqueous layer was extracted with ethyl acetate (3×). The combined organic layers were washed with brine, and concentrated to give a residue, that was purified to give 4-N-substituted-2-O-substituted-5-fluorobenzenecarbonitrile.

4-N-substituted-2-O-substituted-5-fluorobenzenecarbonitrile was reduced with DIBAL-H to give a 4-N-substituted-2-O-substituted-5-fluorobenzaldehyde according to procedure in Scheme 10.

The aldehyde was converted to the corresponding thiosemicarbazone using Scheme 7.

Scheme 13

A solution of 4-N-substituted-2,5-difluorobenzenecarbonitrile (1 equiv.), amine (1.5 equiv.) and DIEA (2 equiv.) in NMP was heated in a Smith Microwave (Personal Chemistry) for 30 minutes. The reaction mixture was purified on silica gel to give 4-N-substituted-2-N-substituted-5-fluorobenzenecarbonitrile.

4-N-substituted-2-N-substituted-5-fluorobenzenecarbonitrile was reduced with DIBAL-H according to procedure described in Scheme 10 to give 4-N-substituted-2-N-substituted-5-fluorobenzaldehyde.

Preparation of amino {3-[5-(3-chlorophenyl)(2-furyl)](2-pyrazolinyl)}methane-1-thione

To a solution of 5-(3-chlorophenyl)furan-2-carbaldehyde (1.0 equiv.) in THF at 0° C. was added MeMgBr in ether (3.0 equiv.) and stirred for 45 min. The reaction was quenched with water, diluted with ether and filtered through Celite. The organic layer was separated and washed with brine, dried over MgSO₄, and concentrated to give the 1-[5-(3-chlorophenyl)-2-furyl]ethan-1-ol.

To a solution of secondary alcohol(1.0 equiv.) in CH₂Cl₂ was added MnO₂ (10 equiv.). The reaction was stirred overnight, filtered through Celite, and concentrated to give 1-[5-(3-chlorophenyl)-2-furyl]ethan-1-one.

To a mixture of ketone (1.0 equiv.), paraformaldehyde (2.0 equiv.), and dimethylamine hydrochloride (2.0 equiv) and molecular sieves in ethanol was added concentrated hydrochloric acid (cat.). The reaction was refluxed overnight under nitrogen and the concentrated. A few drops of HCl was added, and the mixture was worked up with DCM and water. The organic layer was discarded. The aqueous layer was adjusted to basic and extracted with DCM (3×). The organic layer was washed with brine, dried over MgSO₄, and concentrated to yield 3-(dimethylamino)-1-[5-(3-chlorophenyl)(2-furyl)]propan-1-one.

Thiosemicarbazide (1.0 equiv.) was dissolved in MeOH upon heating under nitrogen. Aqueous sodium hydroxide (6 M, 9.0 equiv.) was added to the reaction. A methanol solution of 3-(dimethylamino)-1-[5-(3-chlorophenyl)(2-furyl)]propan-1-one (1.0 equiv) was then added dropwise to the reaction mixture. The solvent was removed and the residue was dissolved in DCM and washed with water, brine, dried over MgSO₄, and concentrated. The final compound was purified by preparative-HPLC to give amino {3-[5-(3-chlorophenyl)(2-furyl)](2-pyrazolinyl)}methane-1-thione; LC/MS m/z 306.2 (MH+); Rt=3.06 minutes.

To a solution of 4-pyridylmethylamine (1.0 equiv.) and triethylamine (2.0 equiv.) in CHCl₃ was added CS₂ (1.0 equiv.)) and stirred overnight. The reaction was cooled to 0° C. and ethyl chloroformate (1.0 equiv.) was added dropwise. The reaction was stirred for 15 min at 0° C. and then stirred at room temperature for 2 hrs followed by addition of (tert-butyl)oxycarbohydrazide (1.2 equiv.). After stirring for an addition hour the mixture was washed with aqueous citric acid (5%), saturated NaHCO₃, brine, dried over MgSO₄, and concentrated. The desired Boc protected thiosemicarbazide was purified using column chromatography.

To a solution of Boc protected thiosemicarbazide (1.0 equiv.) dissolved in DCM was added HCl in dioxane (2M, 8.3 equiv.) and stirred for 15 min. MeOH is then added to dissolve the precipitate, followed by addition of the furfural, and small amount of acetic acid (0.5 mL). The mixture is stirred overnight and the solvents are removed to give a residue purified by preparative-HPLC to give the thiosemicarbazone.

Synthesis of 4-[4-(4-methylpiperazin-1-yl)phenoxymethyl]benzaldehyde

To a solution of 4-piperazin-1-yl phenol (1 equivalent) in CHCl₃, cooled to 0° C., was added di-t-butyl dicarbonate (1 equivalent) in CHCl₃ drop-wise. The solution was stirred at 0° C. for 1 hour before removing from the cold bath and stirring at ambient temperatures for 18 hours. The organic solution was washed aqueous NaHCO₃ and brine dried over MgSO₄ and concentrated the crude material was used without purification.

A solution of the resulting 4-(1-BOC-piperazin-4-yl)phenol (1 equivalent) in dry CH₃CN was slowly added drop-wise to a slurry of NaH (1 equivalent) in dry CH₃CN at room temperature under N₂. The slurry was stirred at room temperature for 2 hours before the solids were filtered and washed with Et₂O.

Sodium 4-(1-BOC-piperazin-4-yl)phenoxide (1 equivalent) and methyl 4-bromomethylbenzoate (1 equivalent) were combined in dry acetone and heated to reflux at 60° C. for 18 hours. The slurry was filtered and the filtrate was then concentrated to provide the crude methyl 4-[4-(1-BOC-piperazin-4-yl)phenoxymethyl]benzoate, that was used without purification.

To a slurry of LiAlH₄ (4 equivalents) in dry THF, cooled to 0° C. under N₂, was slowly added drop-wise a solution of methyl 4-[4-(1-BOC-piperazin-4-yl)phenoxymethyl]benzoate (1 equivalent) in dry THF. Once the addition was complete, the slurry was heated to reflux at 80° C. for 1 hour. The slurry was subsequently cooled to 0° C. and treated with water, 10% aq. NaOH and with water again. The resulting solids were filtered, and the filtrate was diluted with chloroform, washed with brine, dried over MgSO₄ and concentrated, providing the crude 4-[4-(4-methylpiperazin-1-yl)phenoxymethyl]benzyl alcohol that was used without purification.

To a solution of DMSO (2.6 equivalents) in dry DCM, cooled to −78° C. under N₂ was added oxalyl chloride (1.1 equivalents) in DCM drop-wise. The solution was stirred at −78° C. for 5 minutes before a solution of 4-[4-(4-methylpiperazin-1-yl)phenoxymethyl]benzyl alcohol (1 equivalent) in DCM was added drop-wise, and allowed to stir at −78° C. for another 30 minutes. Triethylamine (2.5 equivalents) was slowly dripped in before allowing the solution to reach ambient temperatures. The solution was washed with aqueous NaHCO₃ and brine, dried over MgSO₄ and concentrated to provide the crude 4-[4-(4-methylpiperazin-1-yl)phenoxymethyl]benzaldehyde that was converted to thiosemicarbazones according to Scheme 7. Pyrroles

Preparation of tert-butyl (2E)-3-(2,4-dichlorophenyl)prop-2-enoate (2)

Neat DIC (1.4 eq) was added to a well stirred solution of cinnamate (1 eq), t-butyl alcohol (4 eq), DMAP (1.4 eq) and CH₂Cl₂ under argon at rt. (Note—The cinnamate must be completely in solution that may require gentle warming. Allow the solution to cool to room temperature before adding the DIC. To avoid an exotherm on larger scales, it may be prudent to dilute the DIC with CH₂Cl₂ before the addition and have an ice bath ready.) After stirring for 8 hours, the reaction develops a white precipitate. The reaction may be monitored by TLC eluting with 25% EtOAc/Hexane (R_(f) of product was 0.9). The entire reaction was loaded into a separatory funnel (washing with CH₂Cl₂). The organic mixture was washed with citrate, sat. aq. NaHCO₃, water, and brine. The organic layer was dried (Na₂SO₄), filtered, and concentrated to dryness to give the crude product as an oil. The crude oil was mixed with hexane and stirred for 30 min. The precipitate that forms was filtered over celite and the filtrate was evaporated. The hexane mixture was loaded onto a filter plug of silica and eluted with EtOAc/hexane (97:2 v/v). The first eluted UV active fractions are collected and evaporated to give >99% pure 2 (75-80% yields).

Preparation of tert-butyl 4-(2,4-dichlorophenyl)pyrrole-3-carboxylate (3)

Dry ether was added to NaH (1.5 eq as the oil dispersion) under argon. After decanting off the ether via syringe, the NaH was suspended again with fresh ether under argon. A solution of TOSMIC (1.1 eq) and 2 (1 eq) dissolved in a mixture of ether and DMSO was added dropwise to the stirred suspension of NaH at 0° C. over 20-30 min. The addition was mildly exothermic and evolved gas. After the addition, the reaction was allowed to warm to ambient rt. The progress of the reaction was followed by TLC (25% EtOAc/Hexane, the UV active product was at R_(f)=0.4) and LCMS until done (˜2-3 h). Upon completion, the reaction was carefully quenched with sat. aq. NH₄Cl (added slowly to avoid strong gas evolution and exotherm) and diluted with ether. The layers were separated and the organic phase was washed with sat. aq. NaHCO₃, water, and brine. The crude dark solid can be purified by recrystallization. Best results were achieved either through recrystallization directly from a mixture of hot EtOAc/hexane (1:3 v/v) or by dissolving the crude product in minimal hot EtOAc followed by addition of hexane (˜2 volumes of hexane based on the volume of EtOAc). The hot solutions were allowed to cool to room temperature and age over night. The crystals were first filtered and then washed with hexane giving 99% pure product in 60-70% yield.

Preparation of tert-butyl 4-(2,4-dichlorophenyl)-1-[3-(1,3-dioxobenzo[c]azolin-2-yl)propyl]pyrrole-3-carboxylate (4)

Solid NaH (1.5 eq as the oil dispersion) was added in small portions to a solution of pyrrole 3 (1 eq) and 3-bromopropyl phthalimide (1.2 eq) dissolved in DMF stirred at room temperature and flushed with argon. NOTE—Some gas evolves, but the temperature does not seem to rise above 40-50° C. The reaction was stirred for 1.5 h at room temperature under argon. The reaction was followed by TLC (CH₂Cl₂/acetonitrile (95:5 v/v), the UV active product was at R_(f)=0.5) and LCMS. Upon completion, the reaction was quenched with sat. aq. NH₄Cl (add slowly to avoid strong gas evolution and exotherm). Sat. aq. NaHCO₃ was then added to avoid an emulsion, and the basic organic mixture was extracted with ether. The combined ether layers were washed with sat. aq. NaHCO₃, water, brine, dried Na₂SO₄, filtered, and concentrated to dryness to give the crude product. The crude product was purified by eluting through silica with EtOAc/Hexane (1:4 v/v). The purified product contained some residual 3-bromopropyl phthalimide, that did not interfere with subsequent synthetic steps. The material was taken on and used without further purification. Assume a quantitative yield.

Preparation of tert-butyl 1-(3-aminopropyl)-4-(2,4-dichlorophenyl)pyrrole-3-carboxylate (5)

The Pthalimido Pyrrole 4 (1 eq) was dissolved in ethanol and hydrazine (3 eq) at room temperature under nitrogen. Upon heating to reflux, the reaction generated a white precipitate. Stir at reflux until complete (˜2 h) by TLC (CH₂Cl₂/acetonitrile (95:5 v/v), the UV active product was at R_(f)=0.2) and LCMS. Upon reaching completion, the reaction was allowed to cool to room temperature and the precipitate was vacuum-filtered off using a medium to fine cintered-glass filter. The filtrate was concentrated under reduced pressure to a gummy solid. The crude material was taken up in ethanol/EtOAc (1:1 v/v), stirred and the precipitate was filtered off in the same fashion as before. The filtrate was concentrated under reduced pressure and than dried in vacuo for 10-15 min. This process of adding ethanol/EtOAc, filtering and concentrating was done one more time or as needed to remove the majority of the white precipitate and residual hydrazine. The product was then dried in vacuo overnight. The material was used without further purification. Once dried, the reaction yielded the product as a glass (˜87% yield over 2 steps).

Preparation of tert-butyl 1-{3-[(6-amino-5-nitro(2-pyridyl))amino]propyl}-4-(2,4-dichlorophenyl)pyrrole-3-carboxylate (7)

To the premixed dry reagents, pyrrole 5 (1 eq) and powdered 6-chloro-3-nitro-2-pyridylamine (6) (1.1 eq), was added the DMA followed by Hünig's base (2 eq) sequentially with stirring at rt. The reaction was then heated to 80° C. overnight. The reaction was followed by TLC (EtOAc/hexane (1:1 v/v), the UV active yellow product was at R_(f)=0.25), HPLC and LCMS. Upon completion as judged by HPLC, the reaction was allowed to cool to 70° C. Ethylene diamine (anhydrous) was then added to the reaction to destroy any remaining unreacted chloropyridine 6. After 15 min stirring at 70° C., the reaction was cooled and quenched with the addition of sat. aq. NaHCO₃. The aqueous mixture was extracted with EtOAc, and the combined organic layers were washed with sat. aq. NaHCO₃, water, brine, dried, filtered, and concentrated to dryness to give the crude product as a brown-yellow solid. The crude product was purified by flash chromatography eluted with EtOAc/hexane (4:6 v/v). The purified SnAr adduct 7 was isolated in 58% yield as a yellow solid.

Preparation of 1-{3-[(6-amino-5-nitro(2-pyridyl))amino]propyl}-4-(2,4-dichlorophenyl)pyrrole-3-carboxylic acid (8)

In a vial, TFA (catalytic amount) was added to a stirred mixture of tert-butyl ester pyrrole 7 (1 eq), water (0.1%), and CH₂Cl₂ at rt. The vial stirred at room temperature until done (˜12 h. The reaction was then concentrated under reduced pressure at room temperature and dried in vacuo. The crude residue was dissolved again in CH₂Cl₂ and concentrated under reduced pressure at rt. The material was used in the final coupling step without further purification as the TFA salt.

Preparation of N-((1S)-2-hydroxy-isopropyl)(1-{3-1(6-amino-5-nitro(2-pyridyl))amino]propyl}-4-(2,4-dichlorophenyl)pyrrol-3-yl)carboxamide (9,)

(2S)-(+)-2-Aminopropan-1-ol (1.5 eq) was added to a stirred mixture of acid (8) (1 eq), HBTU (1.5 eq), Hünig's base (2 eq) and DMF (premixed sequentially in this order in a vial) at room temperature under argon. The reaction was stirred for 3-4 h until complete as shown by LCMS and HPLC. The reaction mixture was subsequently diluted with EtOAc, washed with NaHCO₃, and concentrated to afford a powder in a 70% yield.

Nomenclature for the Example compounds was provided using ACD Name version 5.07 software (Nov. 14, 2001) available from Advanced Chemistry Development, Inc. Some of the compounds and starting materials were named using standard IUPAC nomenclature.

The compounds of Table 34 were synthesized following the synthetic methodology described above in the Examples and Schemes, and screened following methods 1 and 2 below. The precursors are readily recognizable by one skilled in the art and are commercially available from Aldrich (Milwaukee, Wis.) or Acros Organics (Pittsburgh, Pa.), among others.

Screening Methods for SMIP/SMIS Compounds

Method 1

Candidate small molecule immuno-potentiators can be identified in vitro. Compounds are screened in vitro for their ability to activate immune cells. One marker of such activation is the induction of cytokine production, for example TNF-α production. Apoptosis inducing small molecules may be identified having this activity. These small molecule immuno-potentiators have potential utility as adjuvants and immuno-therapeutics.

In an assay procedure (High Throughput Screening (HTS)) for small molecule immune potentiators (SMIPs), human peripheral blood mononuclear cells (PBMC), 500,000 per mL in RPMI 1640 medium with 10% FCS, were distributed in 96 well plates (100,000 per well) already containing 5 μM of compound in DMSO. The PBMCs were incubated for 18 h at 37° C. in 5% CO₂. Their ability to produce cytokines in response to the small molecule compounds is determined using a modified sandwich ELISA.

Briefly supernatants from the PBMC cultures were assayed for secreted TNF using a primary plate bound antibody for capture followed by a secondary biotinylated anti-TNF antibody forming a sandwich. The biotinylated second antibody was then detected using streptavidin-Europium and the amount of bound europium was determined by time resolved fluorescence. SMIP compounds were confirmed by their TNF inducing activity that was measured in the assay as increased Europim counts over cells incubated in RPMI medium alone. “Hits” were selected based on their TNF-inducing activity relative to an optimal dose of lipopolysaccaride LPS (1 μg/ml), a strong TNF inducer. The robustness of the assay and low backgrounds allowed for the routine selection of hits with ˜10% of LPS activity that was normally between 5-10× background (cells alone). Selected hits are then subjected to confirmation for their ability to induce cytokines from multiple donors at decreasing concentrations. Those compounds with consistent activity at or below 5 μM are considered confirmed for the purposes of this assay. The assay is readily modified for screening for compounds effective at higher or lower concentrations.

Method 2

Each of the compounds in the above Table 34 elicited TNF-α production in human peripheral blood mononuclear cells. Many of the compounds showed activity at less than 20 μM with respect to production of TNF-α. Many of these compounds showed activity at less than 5 μM with respect to production of TNF-α. Many of these compounds showed activity in the production of TNF-α at less than 1.5 μM.

For this reason, each of the R groups of any of the compounds listed in Table 34 are preferred. Additionally, because of the excellent activity of each of the compounds, each of these compounds is individually preferred and is preferred as a member of a group that includes any or all of the other compounds and each compound is preferred in methods of modulating immunopotentiation and in methods of treating biological conditions associated therewith, for example to be used as a vaccine adjuvant. Each of the compounds is also preferred for use in preparation of medicaments for vaccines, immunopotentiation, reducing tumor growth and in treating biological conditions mediated therefrom.

In additon to the procedure described above, methods of measuring other cytokines (e.g. IL1-beta, IL-12, IL-6, IFN-gamma, IL-10 etc.) are well known in the art and can be used to find active SMIP compounds of the present invention.

Compounds may be useful that cause production of TNF-α at higher concentrations, such as 100 μM, 200 μM or 300 μM in the assays described herein. For example Loxoribine causes useful production of TNF-α at 300 μM (see Pope et al Immunostimulatory Compound 7-Allyl-8-Oxoguanosine (Loxoribine) Induces a Distinct Subset of Murine Cytokines Cellular Immunology 162: 333-339 (1995)).

The subject invention also includes isotopically-labeled antiviral compounds, that are structurally identical to those disclosed above, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into antiviral compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively. Antiviral compounds of the present invention, derivatives thereof, and pharmaceutically acceptable salts of said compounds and of said derivatives that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled antiviral compounds of the present invention, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled antiviral compounds of this invention and derivatives thereof can generally be prepared by carrying out known or referenced procedures and by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

In accordance with the present invention, methods are provided for the administration of an effective amount of a SMIP compound to act as an adjuvant. Also provided are immunogenic compositions comprising a SMIP compound, an antigen, and optionally other adjuvants.

As adjuvants, the SMIP compounds are combined with antigens and delivery systems to form a final immunogenic composition or vaccine product.

As immunotherapeutics, the SMIP compounds are used alone or in combination with other therapies for treatment of SARS.

Those of ordinary skill in the art will recognize that physiologically active antiviral compounds, SMIPs or SMISs that have accessible hydroxy groups are frequently administered in the form of pharmaceutically acceptable esters. The antiviral compounds of this invention can be effectively administered as an ester, formed on the hydroxy groups, just as one skilled in pharmaceutical chemistry would expect. It is possible, as has long been known in pharmaceutical chemistry, to adjust the rate or duration of action of the antiviral compound by appropriate choices of ester groups.

Other compounds that can be used in combination with the therapeutic agents described herein include, pentoxifylline (PTX), methylprednisolone, trimetrexate (Neutrexin), Zadaxin (thymosin alpha 1), optionally substituted 5-aminomethinimino-3-methyl-4-isoxazolecarboxylic acid phenylamides, cyclosporine A (CsA), 6-oxo-1,4,5-thiadiazin[2,3-b]quinazoline, 3-amino-2(1H)-thioxo-4(3H)-quinazolinone, gangciclovir, glycyrrhizin, tetracyclines, aminoglycosides, quinolones, bicyclam (1,4-Bis(1,4,8,11-tetraazacyclotetradec-1-ylmethyl)benzene octahydrochloride dihydrate), rapamycin, wortmannin, enalapril, roquinimex/linomide, inactivin, DNCB, AG7088, 9-aminocamptothecin (CPT-11), loxorobine, bropirimine, Ononase ® (ranpimase), statins, such as: lovastatin—Mevacor®, pravastatin—Pravachol®, simvastatin—Zocor®, fluvastatin—Lescol®, atorvastatin—Lipitor® and rosuvastatin—Crestor®.

As used herein, the term “effective amount” means an amount of antiviral compound of the compositions, kits and methods of the present invention that is capable of treating the symptoms of the described conditions. The specific dose of a compound administered according to this invention will, of course, be determined by the particular circumstances surrounding the case including, for example, the compound administered, the route of administration, the state of being of the patient, and the severity of the condition being treated.

The dose of an antiviral compound of this invention to be administered to a subject is rather widely variable and subject to the judgment of the attending physician. It should be noted that it may be necessary to adjust the dose of a compound when it is administered in the form of a salt, such as a laureate, the salt forming moiety of which has an appreciable molecular weight.

The following dosage amounts and other dosage amounts set forth elsewhere in this description are for an average human subject having a weight of about 65 kg to about 70 kg. The skilled practitioner will readily be able to determine the dosage amount required for a subject whose weight falls outside the 65 kg to 70 kg range, based upon the medical history of the subject and the presence of diseases, e.g., diabetes, in the subject. Calculation of the dosage amount for other forms of the free base form such as salts or hydrates is easily accomplished by performing a simple ratio relative to the molecular weights of the species involved.

In general, the pharmaceutical compositions will include at least one antiviral compound in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water, borate-buffered saline containing trace metals or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, lubricants, fillers, stabilizers, etc. Methods of formulation are well known in the art and are disclosed, for example, in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991) or “Remington: The Science and Practice of Pharmacy,” 20^(th) ed., Lippincott Williams & Wilkins, Baltimore, Md. (2000), incorporated herein by reference.

Pharmaceutical compositions for use within the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art.

Many of the active ingredient antiviral compounds are known to be absorbed from the alimentary tract, and so it is usually preferred to administer a compound orally for reasons of convenience. However, the compounds may equally effectively be administered intravenously, subcutaneously, percutaneously, or as suppositories for absorption by the rectum or vagina, if desired in a given instance. All of the usual types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, troches, suppositories and suspensions. Compositions are formulated to contain a daily dose, or a convenient fraction of daily dose, in a dosage unit, that may be a single tablet or capsule or convenient volume of a liquid.

Capsules are prepared by mixing the compound or compounds with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances such as starch of many different kinds, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders.

Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound or compounds. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are substances such as starch, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidine and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders.

A lubricant is generally necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

Tablet disintegrators are substances that swell when wetted to break up the tablet and release the compound or compounds. They include starches, clays, celluloses, algins and gums, more particularly, corn and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp and carboxymethylcellulose, for example, may be used as well as sodium lauryl sulfate.

Tablets are often coated with sugar as a flavor and sealant, or with film-forming protecting agents to modify the dissolution properties of the tablet. The compounds may also be formulated as chewable tablets, by using relatively large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well-established in the art.

When it is desired to administer a compound as a suppository, the typical bases may be used. Cocoa butter is a traditional suppository base, that may be modified by addition of waxes to raise its melting point slightly. Water-miscible suppository bases comprising, particularly, polyethylene glycols of various molecular weights are in wide use.

The effect of the compounds may be delayed or prolonged by proper formulation. For example, a slowly soluble pellet of the compound may be prepared and incorporated in a tablet or capsule. The technique may be improved by making pellets of several different dissolution rates and filling capsules with a mixture of the pellets. Tablets or capsules may be coated with a film that resists dissolution for a predictable period of time. Even the parenteral preparations may be made long-acting by dissolving or suspending the compound or compounds in oily or emulsified vehicles that allow dispersion slowly in the serum.

The combinations of this invention may be administered in a controlled release formulation such as a slow release or a fast release formulation. Such controlled release formulations of the combination of this invention may be prepared using methods well known to those skilled in the art. The method of administration will be determined by the attendant physician or other person skilled in the art after an evaluation of the subject's condition and requirements.

The term “prodrug” means compounds that are transformed in vivo to yield an antiviral compound of the present invention. The transformation may occur by various mechanisms, such as through hydrolysis in blood. A good discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. The term, “prodrug” also encompasses mutual prodrugs in which one or more antiviral compounds are combined in a single molecule that may then undergo transformation to yield the individual antiviral compounds of the present invention.

For example, if an antiviral compound of the present invention contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (C₁-C₈)alkyl, (C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl), carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl and piperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

Similarly, if an antiviral compound of the present invention comprises an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (C₁-C₆)alkanoyloxymethyl, 1-((C₁-C₆)alkanoyloxy)ethyl, 1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl, (C₁-C₆)alkoxycarbonyloxymethyl, N-(C₁-C₆)alkoxycarbonylaminomethyl, succinoyl, (C₁-C₆)alkanoyl, α-amino(C₁-C₄)alkanoyl, arylacyl and α-aminoacyl, or α-aminoacyl-α-aminoacyl, where each α-aminoacyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate).

If an antiviral compound of the present invention comprises an amine functional group, a prodrug can be formed by the replacement of a hydrogen atom in the amine group with a group such as R^(X)-carbonyl, R^(X)O-carbonyl, NR^(X)R^(X)′-carbonyl where R^(X) and R^(X)′ are each independently ((C₁-C₁₀)alkyl, (C₃-C₇)cycloalkyl, benzyl, or R^(X)-carbonyl is a natural α-aminoacyl or natural α-aminoacyl-natural α-aminoacyl, —C(OH)C(O)OY^(X) wherein (Y^(X) is H, (C₁-C₆)alkyl or benzyl), —C(OY^(X)O)Y^(X1) wherein Y^(X0) is (C₁-C₄)alkyl and Y^(X1) is ((C₁-C₆)alkyl, carboxy(C₁-C₆)alkyl, amino(C₁-C₄)alkyl or mono-N- or di-N,N—(C₁-C₆)alkylaminoalkyl, —C(Y^(X2))Y^(X3) wherein Y^(X2) is H or methyl and Y^(X3) is mono-N- or di-N,N—(C₁-C₆)alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl.

The compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Antiviral, SMIP, SMIS, or other immunomodulating compounds are prepared or obtained as described herein and in the U.S. Patents and published international patent applications listed in Table 1, Table 2, Table 34 and Table 35. The antiviral compounds can be formulated in pharmaceutically acceptable compositions suitable for delivery to the lungs. Particular formulations include dry powders, liquid solutions or suspensions suitable for nebulization and propellant formulations suitable for use in metered dose inhalers. The preparation of such formulations is well know to those skilled in the art, and is described in U.S. Pat. Nos. 5,814,607 and 5,654,007 and in the U.S. Patents and published international patent applications listed in Table 3 the disclosures of which are incorporated herein by reference.

Dry powder formulations will comprise an antiviral compound in a dry, optionally lyophilized form with a particle size within a preferred range for deposition within the lung. Typically the particle size for deposition in the lung will range between 1 and 5 μm. When systemic delivery of the antiviral compound via absorption from the lung into the bloodstream is desired the antiviral compound formulation particle size is generally between 0.1 and 2 μm in size. The preferred size range of particles can be produced using methods such as jet-milling, spray drying and solvent precipitation, for example. Dry powder devices typically require a powder mass in the range from about 1 mg to 100 mg to produce an aerosolized dose. Thus, the antiviral compound will typically be combined with a pharmaceutically acceptable dry bulking powder. Preferred dry bulking powders include sucrose, lactose, trehalose, human serum albumin (HSA), phospholipids and glycine as well as those disclosed in the documents listed in Table 3. Dry powders can be administered to the subject in conventional dry powder inhalers. For liquid formulations the antiviral compound can be dissolved in any recognized physiologically acceptable carrier for use in delivery of aerosolized formulations. Such carriers include buffered and unbuffered aqueous solutions for water soluble compounds, and physiological solutions including saline solution (preferably between 0.2 and 2 N NaCl). For antiviral compounds with limited solubility, other liquid vehicles such as ethanol, propylene glycol and ethanol-propylene combinations may be used. The antiviral compounds may also be administered as solids in suspension.

For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray administered via pressurized packs or a nebulizer, with the use of a propellant, e.g., air, dichlorordifluoromethane, dichloroterafluoroethane or other suitable gas. Preferably, for incorporation into the aerosol propellant, the antiviral compound formulations of the present invention will be processed into respirable particles as described above for the dry powder formulations. The particles are then suspended in the propellant, optionally being coated with a surfactant to enhance their disbursement. In the use of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.

Commercially available jet nebulizers are available and may be used to deliver aerosolized antiviral compound to a subject. Such jet nebulizers include, but are not limited to, those supplied by AeroTech 11 (CIS-US, Bedford, Mass.). In addition, for delivery of aerosolized antiviral compound to the lungs of a subject an oxygen source can be attached to the nebulizer providing a flow rate of, for example, 10 L/min. In general, inhalation is performed over a 5-40 minute time interval through a mouthpiece during spontaneous respiration. The present invention provides for novel compositions comprising a suitable carrier and aerosolized antiviral compound in doses sufficient to reduce or ameliorate viral load and SARS symptoms in subjects having SARS. Such doses can be lower than corresponding systemic doses that may be used to those generally used to reduce or ameliorate viral load and SARS symptoms in subjects having SARS.

The antiviral, SMIP, SMIS, and immunomodulating compositions of the present invention may be administered with a steroidal anti-inflammatory drug for the treatment of SARS and SARS symptoms. Examples of steroidal anti-inflammatory drugs of the invention include hydrocortisone, prednisolone, dexamethasone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, betamethasone, etc.

The antiviral compound composition of the invention is nebulized predominantly into particle sizes allowing a delivery of the drug into the terminal and respiratory bronchioles. For efficacious delivery of antiviral compound to the lung endobronchial space of airways in an aerosol, the formation of aerosol particles having mass medium average diameter predominantly between 1 to 5 μm is necessary. The formulation must additionally provide conditions that would not adversely affect the functionality of the airways. Consequently, the formulation must contain enough of the drug formulated under the conditions that allow its efficacious delivery while avoiding undesirable reaction.

For liquid solutions and suspensions, the choice of the nebulizer is made from among commercially available nebulizers. The jet nebulizers known as Sidestream O, obtained from Medicaid and Pari LCS, LC Plus, and eFlow obtained from Pari Respiratory Equipment, Richmond, Va., are examples of typical nebulizers suitable for the practice of the invention. Ultrasonic nebulizers that produce appropriate particle sizes of about 1 to 5 μm such as Aerosonic by DeVilbiss and UltraAire by Omron are also suitable.

Advantageously, the present invention also provides for a kit for use by a consumer for the treatment and/or prevention of SARS. Such a kit comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of at least one compound from among those described herein or listed in Table 34 and Table 35 or described in the U.S. Patents and published international patent applications listed in Table 1, Table 2, and Table 35 and a pharmaceutically acceptable carrier, vehicle or diluent; (b) a container for holding the pharmaceutical composition; and, optionally, (c) instructions describing a method of using the pharmaceutical compositions for the treatment and or the prevention of SARS. The kit may optionally contain a plurality of antiviral compounds for the treatment of SARS wherein the anti viral compounds are selected from 3C-like protease inhibitors and papain-like protease inhibitors. In a further embodiment, the kit contains an antiviral compound which is an RNA-dependent RNA polymerase inhibitor. When the kit comprises more than one antiviral compound, the antiviral compounds contained in the kit may be optionally combined in the same pharmaceutical composition.

A “kit” as used in the instant application includes a container for containing the separate compositions such as a divided bottle or a divided foil packet. The container can be in any conventional shape or form as known in the art that is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag (for example, to hold a “refill” of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle that is in turn contained within a box.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be packed. Next, the tablets or capsules are placed in the recesses accordingly and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil that is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

It maybe desirable to provide a written memory aid, where the written memory aid is of the type containing information and/or instructions for the physician, pharmacist or subject, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested or a card that contains the same type of information. Another example of such a memory aid is a calendar printed on the card e.g., as follows “First Week, Monday, Tuesday,” . . . etc . . . . “Second Week, Monday, Tuesday, . . . ” etc. Other variations of memory aids will be readily apparent. A “daily dose” can be a single tablet or capsule or several tablets or capsules to be taken on a given day. Also a daily dose of one or more component(s) of the kit can consist of one tablet or capsule while a daily dose of another one or more component(s) of the kit can consist of several tablets or capsules.

Another specific embodiment of a kit is a dispenser designed to dispense the daily doses one at a time in the order of their intended use. Preferably, the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a mechanical counter that indicates the number of daily doses that has been dispensed. Another example of such a memory-aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.

EXAMPLES Example 1 Example of a SARS Virus Isolate

A SARS virus was isolated from clinical specimens of a patient in Frankfurt, Germany (FRA). The isolate was grown in Vero cells. RNA of the SARS virus was extracted and amplified by RT-PCR. Nucleotide sequence of the viral genome was determined by direct sequencing of the PCR product. Computer analysis was used to predict the features of the genome, to compare it to previously known coronaviruses and to the sequence of different SARS virus isolates.

More specifically, isolation and sequence was performed as follows. After the third passage of the SARS virus in Vero cells, viral particles were purified by ultra centrifugation from 3×10⁷ cells supernatant. Viral RNA was extracted by Triazol method (Gibco-BRL). Viral RNA (200 ng) was transcribed into cDNA with avian RNaseH-thermostable reverse transcriptase following the instructions of the manufacturer (ThermoScript RT System, Invitrogen). Briefly, either 50 pmoles of oligo (dT)₂₀ (SEQ ID NO: 7389) or 25 ng of random hexamers were used to prime the RT reaction in a 20 μl final volume. Amplification and sequencing of the SARS genome were accomplished by direct sequencing of PCR products obtained with: i) specific primers from conserved regions of homology found through multiple alignment among known coronaviruses; ii) oligonucleotides designed around short sequences of SARS isolates available on the Web through WHO network laboratories; iii) degenerate primers to amplify the cDNA mixture with multiple overlapping fragments as end products. Gap closure was realized by long distance PCR with high fidelity Taq (Expand High Fidelity system, Roche) using primers designed on selected fragments. Sequence was collected by primer walking using a BigDye terminator chemistry (Applied Biosystems) and an automated DNA sequencer (3700 capillary model, Applied Biosystems). After obtaining a first pass of the entire genome, a set of both forward and reverse primers were used to amplify and sequence de novo the genome using as a template DNA segments of 2 kb on average. Readings from overlapping fragments were automatically assembled by AutoAssembler (Applied Biosystems) and the 29,740 bp contiguous edited manually.

Computer analysis of the sequence was performed as follows. The GCG Wisconsin Package suite (version 10.0) was used for computer analysis of gene and protein sequences. The PSORT program (http://psort.nibb.ac.jp/) was used for localization predictions. For secondary structure analysis, the PHD software available on the Web at http://cubic.bioc.columbia.edu/predictprotein/was applied. The PSI-BLAST algorithm was used for homology searches (http://www.ncbi.nlm.nih.gov/blast) using the non-redundant protein database. ClustalW was applied to obtain multiple sequence alignments of gene and protein sequences. The LeamCoil-VMF program was used to predict coiled-coil regions in the spike proteins (http://learncoil-vmf.lcs.mit.edu/cgi-bin/vmf). Leucine zippers were predicted with the program 2ZIP, available at http://2Zip.molgen.mpg.de.

Phylogenetic analysis was performed using the neighbor-joining algorithm as implemented in the program NEIGHBOR within the Phylogeny Inference Package (Phylip) (Felsenstein J 1993, program distributed by the author). Bootstrap analysis was always performed with 100 replicates using the program Seqboot. Trees were handled and displayed using TreeView. The program HMMER was used to generate sequence profiles from multiple sequence alignments of the S1 domains of spike proteins. Subsequently, the HMMPFAM program was used to compare the S1 domain of SARS spike to the profiles.

The genome of this SARS virus isolate is 29,740 bases long and the overall structure of the genome is similar to that of the three known groups of coronaviruses. Starting from the 5′ end a leader sequence, an untranslated region (UTR) and two overlapping open reading frames coding for one polyprotein containing the enzymes necessary for replication can be identified. They are followed by a region coding for the spike (S), envelope (E), matrix (M), nucleocapsid (N) structural proteins and eight additional ORFs specific for the SARS virus. At the 3′-end of the genome a UTR with a poly(A) is located. The overall homology to coronaviruses groups 1, 2 and 3 is low and therefore the SARS virus belongs to a new group (group 4) of coronavirus. More detailed analysis of the spike protein amino acid sequence shows that the SARS virus isolate is more closely related to coronavirus group 2.

The complete genome sequence of the SARS virus isolate is 29,740 bp in length. The sequence is available on Genbank and has a GC content of 40.8%, comparable with that of known viruses of the same family. Genome structure is similar to that of other coronaviruses. 14 open reading frames have been predicted. The principal features of the genome and gene products are illustrated reported in FIG. 17 and Table 10. The comparison between the SARS genome and those of group 1, 2 and 3 coronaviruses is reported in FIG. 18.

Nucleotides 1-73 contain a predicted RNA leader sequence followed by an untranslated region (UTR) of 197 nucleotides. The UTR is followed by two overlapping open reading frames (ORF1a, ORF1b), which encompass two-thirds of the genome (nucleotides 265-21485). They encode for a large polyprotein, which is predicted to be processed by viral proteases to generate the replicase complex. The 3′ part of the genome contains the genes coding for the four structural proteins (S, spike protein, E, envelope protein, M, matrix glycoprotein, and N, nucleocapsid protein), and eight predicted ORFs of unknown function (FIG. 17). Finally, at the 3′ end of the genome, we found a second UTR of 340 bases followed by a poly(A) tract. We identified a putative intergenic (IG) sequence also referred to as transcription-associated sequence (TAS), which is a typical feature for coronaviruses. The IG sequence is characterized by 6-18 nucleotides present at the 3′ end of the leader and can be found in front of each gene. The IG sequence plays a key role in RNA transcription and its regulation. The IG sequence of the SARS virus is characterized by the sequence SEQ ID NO: 7293 and is present nine times in the genome (FIG. 17). The sequence of the leader and IG are peculiar for each coronavirus and represent a specific signature for the virus.

The Replicase Region

The replicase gene, ORF1ab (SEQ ID NO: 7232), consists of two overlapping ORFs, ORF1a and ORF1b, which can be translated as a single polyprotein by frame shift of the ribosome in position 13,393, within the polymerase encoding region. See Brierley et al, Embo J 1987: 6(12): 3779-3785. As expected, a stem-loop sequence is present ten base pairs downstream of this site (SEQ ID NO: 7390; 5′-CGGTGTAAGTGCAGCCCGTCTTACACCG-3′). The polyprotein is cleaved co- and/or post-translationally into multiple proteins by its own encoded proteases. Using the cleavage consensus sequence and by analogy with other coronaviruses, we have mapped the possible cleavage sites of the polyprotein and have identified 14 products, which comprise the leader protein p28, the homologue of the MHV p65 protein and other twelve proteins, named from nsp1 to nsp13 (nsp, non structural protein) (FIG. 17 and Table 10). The amino acid sequence analysis suggests the presence of several functional motifs within the putative ORF1ab proteins. In particular, we have mapped two potential proteases (nsp1 and nsp2), one growth factor-like motif (nsp7) within ORF1a, whereas in ORF1b we identified the RNA polymerase (nsp9), and a predicted helicase (nsp10). The other predicted cleavage products (nsp3, nsp4, nsp5, nsp6, nsp11, nsp12 and nsp13) are proteins of unknown function. Many of these proteins are presumably present in the RNA replication complex, which is associated with the membranous structures in the infected cells. In particular, nsp3 and nsp4 contain hydrophobic domains. As shown in FIG. 18, the replicase region of SARS has a similar organization to group 1, 2 and 3 coronaviruses; however, the overall aminoacid conservation is low (Table 11). The most conserved proteins are the polymerase and the helices.

Nsp1 is the papain-like cysteine protease (PLP), which cleaves the first two protein products (leader protein p28 and p65 homologue). Within the nsp1 of MHV, two domains with papain-like protease activity (PLP1 and PLP2) have been mapped, (Kanjanahaluethai et al (2000) J. Virol 74(17):7911-21) which are also conserved with Bovine, transmittable gastroenteritis virus (TGV) and Human 229E coronaviruses. However, by sequence alignment with the SARS nsp 1, we identified only one PLP domain containing the catalytic residues Cys833 and His994.

Nsp2 is the chymotrypsin-picornavirus 3C-like protease (3CLp), which is responsible for the post-translational processing of the other 12 proteins, most of them cleaved at Q/A or Q/S sites. (Ziebuhr et al (1999) J. Virol 73(1):177-85). It also performs autoproteolytic activity. The principal catalytic residues are well conserved with other coronaviruses and are located at position His41 and Cys145. Furthermore, even the conserved aminoacids Tyr161 and His163, which are believed to be involved in substrate recognition and to be indispensable for proteolytic activity, (Hegyi et al (2002) J. Gen Virol 83(Pt3):581-593) were found in the sequence of the SARS 3CLp.

The invention includes the orflab sequence of SEQ ID NO: 9960 and the orfla sequence of SEQ ID NO: 9961, including fragments, variants, homologs, etc. thereof.

The Structural Region

Analysis of the nucleotide sequence at the 3′ part of the SARS genome identified 12 predicted open reading frames. They are coded within 8.2 kb and comprise the four structural proteins S, E, M and N, common for all coronaviruses and eight predicted ORFs, which are specific for this virus (FIG. 18). SARS-specific IG sequences upstream of most ORFs (FIGS. 17 & 18) suggest that most genes are likely to be transcribed independently. Interestingly, sequences identical to the group 2 IG are also present at the end of the RNA leader and in front of the Matrix encoding gene and of ORF 10.

The spike is a type I glycoprotein, which forms the large spikes on the surface of the virion and is responsible for receptor-binding and membrane fusion. (Gallagher (2001) Adv Exp Med Biol 494: 183-92). The protein is 1255 residues long with 17 predicted N-glycosylation sites. It has a 13aa leader peptide and a 17 aa C-terminal membrane anchoring sequence (1202-1218). Some (MHV, HCoV-OC43, AIBV and BCoV), but not all (TGV, FIPV, HCoV-229E) coronavirus spike proteins are proteolytically cleaved in two subunits, S1 and S2. S1 is supposed to form the bulbous head, which stays non-covalently linked to the C-terminal membrane anchor. Cleavage is mediated by a basic aminoacid sequence, which resembles the consensus sequence for a furin cleavage site. (Garten et al., Biochimie 1994; 76(3-4): 217-225). However, in case of this SARS virus isolate, we were not able to identify such a sequence, implicating that the S protein of this SARS virus isolate is unlikely to be cleaved during maturation. Secondary structure predictions indicated that the global architecture of the spike protein is conserved within all known coronaviruses. The S1 domain is mainly formed by beta sheets and likely adopts a globular fold, while in the S2 domain extensive alpha helical regions are predicted. In addition, the LearnCoil-VMF program, specifically designed to identify coiled-coil-like regions in viral membrane-fusion proteins, predicts two coiled-coils within S2, spanning aminoacids 900-1005 and 1151-1185, respectively (FIG. 19). Both coiled-coil regions contain a leucine-zipper motif, which is also present in the spikes of all coronaviruses. Leucine zippers are known to promote protein oligomerization; since the spike proteins of TGV and MHV form hetero-trimers, (Delmas et al, J Virol 1990; 64(11):5367-5375) (Godeke, et al., J Virology 2000; 74(3):1566-1571) it is conceivable that in SARS leucine zippers play a role in promoting and/or stabilizing a similar quaternary structure. The spike protein plays a major role in the biology of coronaviruses because the S1 domain contains the receptor-binding domain and the virus neutralizing epitopes, while the S2 domain is involved in the process of membrane fusion, which is essential for virus infectivity. As expected, multiple sequence alignment of different spike proteins showed a major degree of variability within the S1 domain, whereas S2 is more conserved.

The envelope protein E is a very short polypeptide of 76 aa, involved in the morphogenesis of the virion envelope. (Godet et al., Virology 1992; 188(2):666-675). Computer analysis predicts a long transmembrane domain close to the N-terminus and two N-glycosylation sites. The level of aminoacid similarity with other coronaviruses is very low and the best homology is with the small envelope protein of the transmissible gastroenteritis virus (TGV).

The matrix glycoprotein (M) is a 221-residue polypeptide with a predicted molecular weight of 25 kDa. Computer analysis predicts a topology consisting of a short aminoterminal ectodomain, three transmembrane segments and a carboxyl terminus located at the interior side of the viral envelope. In analogy with the matrix glycoprotein of TGV, that of the avian infective bronchitis virus (AIBV) and that of the hypervirulent MHV-2 strain the SARS M glycoprotein is N-glycosylated at the N-terminus. SARS M protein shows highest similarity to group 2 viruses (Table 11).

Finally, the nucleocapsid protein N is a 397-residue-long phosphoprotein that interacts with viral genomic RNA to form the nucleocapsid. The level of conservation with other coronaviruses is low, ranging from 26.9% identity with the HCoV-229E to 37.4% identity to the Bovine coronavirus (BcoV) (Table 11). Epitope analysis of the nucleocapsid protein has been carried out (Li et al. (2003) Geno Prot & Bioinfo 1:198-206) in which the epitope site at the C terminus of the protein was located as SEQ ID NO: 7394 (amino acids 371-407 of SEQ ID NO: 6052).

In addition to the above fundamental proteins, many viruses express a set of other peptides, which are generally dispensable for viability, but can influence the infectivity potential of the virus. (de Haan et al., Virology 2002; 296(1):177-189). These proteins are generally conserved within members of the same serogroup, but differ profoundly among the groups. For this reason, they are generally referred to as group-specific proteins (FIG. 11). Members of the group 1, represented here by HcoV-229E, have two group-specific genes located between the S and E genes and sometimes one or two ORFs downstream of the N gene, preceding the 3′ UTR region of the genome. Viruses of the group 2, with MHV as prototype, have two group-specific genes (2a and HE) between ORF1b and S, as well as other two between S and E genes. Finally, the group 3 viruses, represented by the prototype AIBV, have two group-specific genes between S and E and other two between the M and N genes.

With the exception of the hemagglutinin esterase HE, for which hemagglutinating and acetyl-esterase enzymatic activities have been demonstrated, all the other group-specific ORFs encode proteins whose role has not yet been established.

Interestingly, the arrangement of specific genes in the SARS genome is peculiar and the predicted ORFs do not display any significant homology with ORFs present in the other coronaviruses, nor with any other known protein from different organisms. Like viruses of the group 1 and 3, SARS lacks the HE hemagglutinin and does not contain ORFs between the ORF1b and the S gene. Furthermore, two predicted ORFs (ORF3 and ORF4) are encoded in the region between S and E, and superimpose for most of their length. ORF3 has an IG sequence 2 bp upstream of the ATG start codon. In contrast to the other groups, SARS contains five predicted ORFs in the region between M and N genes. ORF7 is located 10 bases downstream of the stop codon of M gene, and has an IG sequence 155 nucleotides upstream from the ATG start codon. Similarly, ORF8 and ORF10 present an IG right upstream of their ATG start codons. On the other hand, the 5′ ends of ORF9 and ORF11 shortly superimpose with the flanking genes, and for this reason they do not need an IG to activate transcription. ORF12 totally superimposes with the N gene and shares very low homology with a 22 kDa protein of the MHV virus, coded in the corresponding region.

Despite the absence of indications of possible localization and function deriving from sequence similarity, ORF3, ORF7 and ORF8 contain hydrophobic segments, suggesting association with membrane structures. In addition, ORF3, the longest among the SARS specific gene, is the only one that encodes for a peptide containing a high number of predicted O-glycosylation sites (Table 11). Predicted N-glycosylation sites have been identified in ORF3, ORF11 and ORF12.

Two shorter ORFs in the non-structural regions are SEQ ID NOS: 9965 and 9966. The invention includes polypeptides with these sequences, and also fragments, variants, etc.

Phylozenetic Analysis

The substitution frequency within 922 conserved bases from the pol gene of eleven coronaviruses from the three different serogroups has been used in the past to show that the variability within members of each serogroup is much smaller than between members of different serogroups, confirming the previously described serological groupings. (Stephensen et al., Virus Res 1999; 60(2):181-9). We used the 922 bp region of the pol gene of SARS and aligned it with the same fragment from other 12 coronaviruses. The tree obtained showed that the SARS virus is distinct from the other three groups of coronaviruses (FIG. 20). Similar results were obtained using the full-length aminoacid sequences of pol, 3CL-protease and helicase from the replicase region and those of the spike and the matrix glycoproteins from the structural region (data not shown). These data confirmed that the entire genome of the SARS virus clusters in a new group (group 4) of coronavirus.

To gain more resolution for possible evolutionary relationships we performed the analysis using consensus sequences of predicted domains of the proteins. In particular, we generated consensus sequences of the S1 domain of the spike protein from the group 1 and group 2 and then we compared them to the S1 domain of the SARS spike. No consensus could be generated from the group 3 since only the spike protein of AIBV is known. Interestingly, the tree constructed from the alignment of SARS SI with the consensus generated from the two groups of spike proteins was different from that in FIG. 20, and showed a much closer relationship between SARS and group 2 coronaviruses (FIG. 21A). Further analysis showed that 19 out of the 20 cysteines present in the SARS S 1 domain are spatially conserved with the group 2 consensus sequence, while only five are maintained either within the group 1 and group 3 sequences (FIG. 21B). Given the fundamental role played by cysteines in protein folding, it is likely that the S1 domain of SARS and group 2 coronaviruses share a similar spatial organization.

Seguence Variability Between SARS Coronaviruses

We compared the FRA sequence to the four complete SARS genomes available on the Web. A total of 30 mutations were detected. Nine of these mutations were silent while 21 resulted in aminoacid substitutions (Table 12). Within ORF1a, three silent and seven productive mutations were detected. In ORF1b, there were five silent and three productive mutations. One of the productive mutations was caused by two nucleotide substitutions resulting in a single aminoacid change. Five changes were located in the spike protein, four of these were productive and one silent. Two productive mutations were in ORF3 and in the matrix glycoprotein M. One productive mutation each was in ORF10 and in the nucleocapsid protein N.

The overall difference between FRA and TOR2 was of nine nucleotides resulting in two silent mutations and seven aminoacid changes. The difference between FRA and Urbani is 12 nucleotides, which result in five silent mutations and seven aninoacid changes. For CUHK 16 nucleotides were different, five of which were silent mutations. For FRA and HKU 14 nucleotide changes resulted in four silent and nine productive mutations.

Example 2 Production, Inactivation and Purification of Whole SARS Virus Using MCS Chromatography Resin Purification Followed by Density Gradient Ultracentrifugation

A SARS isolate FRAI (EMBL: AY310120) was passaged on VERO cells that were cultivated in DMEM (Gibco: Cat No. 21969-035, Lot No. 3078864), Penicillin/Strep (Gibco: Cat No. 15070-063, Lot No. 1120042), and 3% FCS (Gibco: Cat No. 10270-106, Lot No. 40F6130K) at 37° C., 5% CO₂. Trypsin (Gibco: Cat No. 25300-054, Lot No. 3078729) was used for detaching the cells.

For virus production the third passage was used for inoculation of VERO cells at a moi of ˜0.1. Cells were incubated with the virus for 1 h at 37° C. in infection medium (DMEM without PS, FCS); after 1 h cells were washed twice and further incubated at 37° C. for 48 h in the presents of 3% FCS and antibiotics. The supernatant was harvested 48 hours post infection (p.i.) and precleared by centrifugation at 3000 rpm at 4° C. for 10 min.

The SARS virus was inactivated by (β-propiolactone (BPL) treatment (1:2000) for 18 h at 4° C., followed by 3 h at 37° C. Testing the virus on successful inactivation, VERO cells were incubated with 10 ml BPL treated supernatant for 4 days at 37° C.; subsequentially, the supernatant was transferred to a fresh VERO cell culture and further incubated for another 4 days. Cells were checked for cytopathic effect (CPE).

200 ml of the BPL-inactivated SARS virus harvest was then clarified using a 0.65 μm-pore-size filter (47 mm diameter) to pass virus particles and retain cell debris. The filter unit was connected to a Masterflex pump, which accomplished a consistent flow rate of 40 ml/min.

A. MCS Chromatography Purification Step

The filtered virus suspension was then subjected to MCS chromatography. The MCS column was prepared as follows. 27 ml slurry led to 14 ml sedimentated resin which Was packed using a Gotec Superformance Column (diameter 1.0 cm, height 15.7 cm, volume 12.33 ml). 1% of the column volume of a 1% acetone solution was injected to the column and the column was run with a flow of 100 cm/h. The HETP, N and A, values were then calculated as HETP: 0.056 cm, N/m: 1790 and A_(s)=1.20.

The amount of proteins in the purified solution after the MCS chromatography step were assessed with a bicinchoninic acid (BCA) method (Interchim) (see, e.g., http://www.piercenet.com/files/bca.pdj) and electrophoresis.

SDS-PAGE was done in accordance to Laemmli, Nature (1970) 227:680-685. Samples for SDS-PAGE were diluted to a protein concentration of 77 μg/ml. Different protein concentrations were loaded depending on the gel types used (10/12/15 Wells, Novex/Invitrogen): Protein Concentration Number of Wells in the Dilution Load Protein/Well 10 Wells 77 μg/ml 20 μl 1 μg 12 Wells 77 μg/ml 15-20 μl 0.75-1 μg 15 Wells 77 μg/ml 10 μl 0.5 μg

Samples for use in a reducing SDS-PAGE were prepared as, follows: 26 μl sample or diluted sample +10 μl NuPage Sample Buffer (4×) SDS NP0003 +4 μl TCEP Bondbreaker Solution 77720 (1:2 in MilliQ water) Final Volume: 40 μl

The samples were heated for 10 minutes at 70° C. or left at room temperature for 1 hour (leaving the samples at room temperature prevents the M protein of Corona Virus to coagulate/forming complexes), and then centrifuged for approximately one minute at 14,000 rpm in a table top centrifuge.

Markers for use on the gel were prepared as follows. Gel bands containing less than 1 μg of proteins were easily visualised with the silver staining procedure using the Silver Staining Kit Protein, Plus One Staining Protocol (Pharmacia Biotech).

Western blotting was performed as follows. A semi-dry blotting technique was used to transfer the proteins from the SDS gel to a nitrocellulose membrane. The transfer was performed with a current of 0.8 mA/cm² for 1 hour. A rabbit polyclonal antibody against SARS virus was used to perform the immuno probing using the Western Breeze, Novex Chromogenic Western Blot Immunodetection Kit (Novex/Invitrogen).

The chromatogram of the inactivated SARS MCS capture step is depicted in FIG. 27. To estimate purity, MCS chromatography fractions were analysed by silver staining on NuPage 10% or 4-12% Bis-Tris-Gel (Novex) under reduced conditions, heated for 10 minutes at 70° C. (FIG. 28). THe fractions were also analysed under the same conditions by western blot (FIG. 29) to estimate using PAK 11/03 SARS Cov 270603 neutralizing titer 1:512 (this antibody was used for this and subsequent western blots). Purity estimates are as follows: Volume/ [Protein]/ Total Protein/ Step Recovery Sample ml μg/ml mg Protein/% Corona Harvest 100 2547.6 254.76 100 After 100 2440.3 244.03 95.8 Filtration = Load Flow Through 85 2321.4 197.32 77.5 Wash 49.32 468.5 23.11 9.1 Peak 1 12.12 252.7 3.062 1.2 Total Recovery — — 464.4 86.5 B. Density Gradient Ultracentrifugation Step

The eluted SARS virus fraction was then subjected to density gradient ultracentrifugation with a swinging bucket rotor to further purify the inactivated virus. 3 ml of MCS peak fraction were loaded onto a linear gradient (15-60% sucrose; 17 ml 15% and 17 ml 60% sucrose in gradient mixer). The separation was performed with a Beckman SW 28 rotor at 20,000 rpm for 2 hours.

The content of sucrose and protein in the linear density gradient ultracentrifugation fractions are depicted in the following table, the graph in FIG. 30 and the estimation of purity in FIG. 31: Fraction Fraction Size/ml [Sucrose]/% [Protein]/μg/ml 1 2 61 96.12 2 2 59.4 98.62 3 2 57.5 87.63 4 2 54.5 86.91 5 2 50.5 79.9 6 2 47.2 74.3 7 2 43.7 68.05 8 2 40.2 60.43 9 2 37.2 57.38 10 2 34 53.12 11 2 30 50.63 12 2 25.7 35.02 13 2 22.4 35.33 14 2 19.5 39.25 15 2 15.5 69.79 16 2 8.5 169.03 17 2 8.5 128.96

The protein concentration of fraction 11 (FIG. 31 SDS-gel) was measured again against a standard curve prepared in 30% sucrose and lead to a protein concentration of 3.67 μg/ml (0.05 μg on the gel). The M protein appears to be missing in this preparation possibily due to sample treatment procedure (heated samples).

There may be discrepancies in the protein concentration measurements in Table 2 due to sucrose interface with this assay.

Example 3 Production, Inactivation and Purification of Whole SARS Virus Using MCS Chromatography Resin Purification Followed by Density Gradient Ultracentrifugation

Inactivated SARS virus was prepared as described in Example above.

A. MCS Chromatography Purification Step

In this example, 200 ml of inactivated SARS virus harvest were subjected to MCS chromatography. The chromatogram of the capture step of inactivated SARS virus purification with MCS is depicted in FIG. 32, the protein recovery in the following table and the estimation of purity in FIG. 33: Volume/ [Protein]/ Total Protein/ Step Recovery Sample ml μg/ml mg Protein/% Corona 200 2239.2 447.83 100 Virus Harvest After 200 2245.1 449.02 100.3 Filtration = Load Flow Through 185 2126.3 393.37 87.8 Wash 49.32 450.1 22.2 5.0 Peak 1 4.43 1245.6 5.52 1.2 Total Recovery — 421.08 93.7 Density Gradient Ultracentrifugation Step

3.5 ml of MCS peak fraction were then loaded onto a linear gradient (15-40% sucrose: 16 ml 15% and 16 ml 40% sucrose in gradient mixer). The separation was performed with a Beckman SW 28 rotor at 20,000 rpm for 2 hours.

The content of sucrose and protein in the linear density gradient ultracentrifugation fractions are depicted in the following table and the graph in FIG. 34: Tube Fraction Size/ml [Sucrose]/% [Protein]/μg/ml 1 2 40 45.86 2 2 39 45.68 3 2 37.5 44.14 4 2 35.5 37.82 5 2 33.5 34.48 6 2 31.5 31.76 7 2 30.5 29.49 8 2 28 30.87 9 2 25.5 31.7 10 2 23.5 26.74 11 2 21.75 23.58 12 2 20 35.33 13 2 18 96.38 14 2 14.5 523.79 15 2 8 941.97 16 2 8 696.7

Protein recovery is shown in the following table and the estimation of purity is shown in FIG. 35. Electron Micrograph pictures of density gradient fractions 8, 9 and 10 are shown in FIG. 36: Protein/ Total Step Step Volume/ml μg/ml Protein/mg Protein % Load 3.5 ml 1245.6 4359.6 100 Bulk Protein Fractions 3.5 ml 720.8 4324.9 99.2 Viral Peak Fraction   8 ml 29.7 237.6 5.5 Total Recovery 4562.5 104.7

Example 4 Mouse Immunization with Inactivated SARS Virus

Mice were immunized subcutaneously on days 0, 14, and 28 with 5 μg BPL-inactivated SARS-CoV particles (BPL-SARS-CoV), either alone or together with Alum or MF59 as adjuvants. Serum was collected on days 0 (pre-immunization), 13 (post 1st immunization), 28 (post 2nd), and 35 (1 week post 3rd immunization). Neutralizing antibodies were assessed for blocking SARS-CoV infection of Vero cells in vitro. After 3 immunizations, neutralization titers were in the range 1:100-1:1000, which are levels similar to those present in the serum of SARS convalescent patients. As shown in the following table, the non-adjuvanted vaccine induced neutralizing antibody after the third immunization, and potency of this vaccine was enhanced significantly by including the adjuvants, with neutralizing antibody appearing after then 2nd immunization and overall titers increasing after then 3rd immunization: Neutralization Titer Immunogen pre post 1st post 2nd post 3rd BPL-SARS-CoV + MF59 (5 μg) <1:20 <1:20  1:158  1:630 BPL-SARS-CoV + Alum (5 μg) <1:20 <1:20 1:67  1:612 BPL-SARS-CoV (5 μg) <1:20 <1:20 <1:20   1:71 PBS <1:20 <1:20 <1:20   <1:20  

Example 5 Balb/cMouse Immunization with Inactivated SARS Virus

A Balb/c mouse model for SARS infection has been developed (Subbarao et al. (2004), J. Virol., 78:3572-77. In this model, Balb/c mice are inoculated intranasally with 10⁴ TCID₅₀ of virus. At 48 hours post-inoculation, a 2-log increase in the TCID₅₀ virus titer can be detected in the lungs of infected mice. While virus replication is readily detected, the mice do not show any SARS disease symptoms and spontaneously clear the virus one week after inoculation. A decrease in virus titer in previously-immunized animals as compared to control animals demonstrates a protective effect of the vaccine being evaluated.

In this example, four Balb/c mice per group are immunized three times with 5 μg BPL inactivated SARS-CoV (days 0, 14, 28) either alone or in combination with MF59 and challenged with 10⁴ TCID₅₀ of SARS-CoV on day 43. Two days following virus challenge the mice are euthanized and SARS-CoV is quantified from nasal turbinates (NT) and lungs and the mean virus titer for each mouse is measured. Control groups received PBS alone, or an influenza virus vaccine (FLU) with or without MF59 adjuvant. Data were as follows (see also FIG. 51), where four mice were tested per group and virus titers are expressed as log₁₀ TCID₅₀ per gram of tissue: Virus replication Virus replication in nasal in lungs of turbinates of challenged mice challenged mice # infected/ Mean (± SE) # infected/ Mean (± SE) Immunogen # tested virus titer # tested virus titer PBS 4/4 6.3 ± 0.3  3/4 2.8 ± 0.35 MF-59 alone 4/4 6.1 ± 0.13 3/4 3.0 ± 0.38 FLU vaccine (5 μg) 4/4 6.3 ± 0.07 3/4 2.9 ± 0.36 FLU vaccine (5 μg) + MF-59 4/4 6.0 ± 0.19 4/4 3.0 ± 0.11 BPL-SARS-CoV (5 μg) 1/4  1.6 ± 0.13* 0/4 Not detected** BPL-SARS-CoV (5 μg) + MF-59 0/4 Not detected* 0/4 Not detected** Two-tailed Student's t-test, compared to PBS-immunized mice, showed: *P < 0.00001 or **P = 0.025

As shown, virus could not be detected in the BPL-SARS-CoV immunized mice. The lower limit of detection of infectious virus in a 10% w/v suspension of lung homogenate was 1.5 log₁₀TCID₅₀/gm, and in a 5% w/v suspension of nasal turbinates the limit was 1.8 log₁₀TCID₅₀/gm. Viral titers in the immunized mammals were thus below these threshold values.

Thus the inactivated SARS-CoV vaccine was very efficient at preventing virus infection, as only one of eight mice immunized with the vaccine, either with or without MF59 adjuvant, was infected. Similar protection was not observed in control groups of PBS diluent, MF59 adjuvant, or influenza virus vaccine with or without adjuvant.

Neutralization titers of sera taken from the animals in the challenge study were assessed at two weeks post-1st, one week post-2nd, and one week post-3rd immunization. Mice immunized with the vaccine with MF59 adjuvant had already developed a neutralization titer of 1:71 after the 2nd immunization, which increased to 1:588 after the 3rd immunization, whereas mice receiving the unadjuvanted vaccine did not have any neutralizing activity post-2nd and a neutralization titer of 1:64 post-3rd immunization. Sera from mice in each of the control groups did not show any neutralization activity. These data clearly demonstrate not only the ability of the inactivated SARS-CoV vaccine to induce protective levels of SARS neutralizing antibodies, but also a beneficial effect of formulating the vaccine with adjuvant for elevated neutralization titers.

Example 6 Preparation of OMV Comprising SARS Viral Antigens

E. coli were transfected with a plasmid of interest (encoding a SARS viral antigen). Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at either 30° C. or 37° C. until the OD₅₅₀ reached 0.6-0.8. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8 000×g for 15 minutes at 4° C. and resuspended in 20 ml of 20 mM Tris-HCl (pH 7.5) and complete protease inhibitors (Boehringer-Mannheim™). All subsequent procedures were performed at 4° C. or on ice.

Cells were disrupted by sonication using a Branson Sonifier 450 and centrifuged at 5 000×g for 20 min to sediment unbroken cells and inclusion bodies. The supernatant, containing membranes and cellular debris, was centrifuged at 50000 g (Beckman Ti50, 29 000 rpm) for 75 min, washed with 20 mM Bis-tris propane (pH 6.5), 1.0 M NaCl, 10% (v/v) glycerol and sedimented again at 50000 g for 75 minutes. The pellet was resuspended in 20 mM Tris-HCl (pH 7.5), 2.0% (v/v) Sarkosyl, complete protease inhibitor (1.0 mM EDTA, final concentration) and incubated for 20 minutes to dissolve inner membrane. Cellular debris was pelleted by centrifugation at 5000 g for 10 min and the supernatant centrifuged at 75000 g for 75 minutes (Beckman Ti50, 33000 rpm). Outer membrane vesicles were washed with 20 mM Tris-HCl (pH 7.5) and centrifuged at 75 000×g for 75 minutes or overnight. The OMV was finally resuspended in 500 μl of 20 mM Tris-HCl (pH 7.5), 10% v/v glycerol. Protein concentration was estimated by standard Bradford Assay (Bio-Rad), while protein concentration of inner membrane fraction was determined with the DC protein assay (Bio-Rad). Various fractions from the isolation procedure were assayed by SDS-PAGE.

Example 7 Immunogenicity, Dose and Route Schedule for Recombinant Spike Protein in Mice

The immunogenicity, route and dosing of the recombinant spike proteins of the invention in mice may be assessed using the below detailed protocol. Preferably, the administered antigen will elicit neutralizing antibody titers at least in the range of 1/100- 1/1000. Increasing doses of antigen can be tested in the range from 5 to 20 μg of recombinant Spike antigen alone or mixed with an equal volume of MF59-citrate, administered SC or IM to anesthetized mice in 100 μl of inoculum. Groups of BALB/c mice, 6 per treatment are primed at day 0 and boosted at day 14 and 28. Group Treatment Dose/Route Sampling interval Number of mice 1-3 Rec-Spike protein 20, 10, 5 μg/SC 7, 21, 35, 42 d 6 per dose level 4-6 Rec-Spike protein 20, 10, 5 μg/SC 7 6 per dose level 7-9 Rec-Spike protein 20, 10, 5 μg/IM 7, 21, 35, 42 d 6 per dose level 10-12 Rec-Spike protein 20, 10, 5 μg/IM 7 6 per dose level 13-15 Rec-Spike - MF59 20, 10, 5 μg/SC 7, 21, 35, 42 d 6 per dose level 16-18 Rec-Spike - MF59 20, 10, 5 μg/SC 7 6 per dose level 19-21 Rec-Spike - MF59 20, 10, 5 μg/IM 7, 21, 35, 42 d 6 per dose level 22-24 Rec-Spike - MF59 20, 10, 5 μg/IM 7 6 per dose level 25 MF59 NA/SC 7, 21, 35, 42 d 6 + 6 (sac d 7 and 42) 27 MF59 NA/IM 7, 21, 35, 42 d 6 + 6 (sac d 7 and 42) 29 Saline NA/SC 7, 21, 35, 42 d 6 + 6 (sac d 7 and 42) 31 Saline NA/IM 7, 21, 35, 42 d 6 + 6 (sac d 7 and 42)

This protocol can also be used to assess the Th1/Th2 profile of the specific immune response elicited by the recombinant Spike protein. Neutralizing and Spike-specific antibody titers will be assessed at days 7, 21, and 35; IgG2a vs IgG1 isotype of the Spike-specific antibodies will be determined at days 21 and 35; in vitro proliferation of lymph node and splenic T cells against the recombinant Spike protein will be determined at days 7 and 42, respectively; IFN-γ and IL-4 production by splenic T cell against the recombinant Spike protein from SARS-CoV will be assessed at day 42. Peripheral blood will be collected at days 7, 21, 35; lymph nodes cells at day 7, and spleen cells at day 42. Neutralizing and Spike-specific antibody titers and isotypes will be determined by inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively. Proliferation of lymph node and splenic cells will be determined by ³[H]-Thymidine uptake. Frequencies of splenic IFN-γ and IL-4 producing T lymphocytes, will be determined by ELISPOT and FACS.

Example 8 Immunogenicity, Dosing and Route Schedule for Spike Proteins in Rabbits

The immunogenicity, route and dosing of the recombinant spike proteins of the invention in rabbits may be assessed using the below detailed protocol. Increasing doses can be tested in the range from 5 to 40 μg of recombinant Spike antigen alone or mixed with an equal volume of MF59-citrate, administered SC or IM to anesthetized animals in 200 μl of inoculum. Groups of New Zealand white female rabbits, 10 per treatment, will be immunized as shown in the table 20 below. The animals will be primed at day 0 and boosted at days 14 and 28. Peripheral blood will be collected at days 7, 21, and 35. Neutralizing and Spike-specific antibody titers will be determined by inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively. Group Treatment Dose/Route Sampling interval Number of rabbits 1-4 Full-length Spike protein 40, 20, 10, 5 μg/SC 7, 21, 35 d 10 per dose level 5-8 Full-length Spike protein 40, 20, 10, 5 μg/IM 7, 21, 35 d 10 per dose level  9-12 Truncated Spike protein 40, 20, 10, 5 μg/SC 7, 21, 35 d 10 per dose level 13-16 Truncated Spike protein 40, 20, 10, 5 μg/IM 7, 21, 35 d 10 per dose level 17-20 Full-length Spike protein - MF59 40, 20, 10, 5 μg/SC 7, 21, 35 d 10 per dose level 21-24 Full-length Spike protein - MF59 40, 20, 10, 5 μg/IM 7, 21, 35 d 10 per dose level 25-28 Truncated Spike protein - MF59 40, 20, 10, 5 μg/SC 7, 21, 35 d 10 per dose level 29-32 Truncated Spike protein - MF59 40, 20, 10, 5 μg/IM 7, 21, 35 d 10 per dose level 33 MF59 NA/SC 7, 21, 35 d 10 34 MF59 NA/IM 7, 21, 35 d 10 35 Saline NA/SC 7, 21, 35 d 10 36 Saline NA/IM 7, 21, 35 d 10

Example 9 Immunogenicity and Dose Schedule for Recombinant Spike in Ferrets

The immunogenicity and dosing of the recombinant spike proteins of the invention in ferrets may be assessed using the below detailed protocol. Three groups of ferrets, 6 for treatment, will be immunized with recombinant SARS-CoV Spike protein from CHO cell lines, alone or mixed with an equal volume of MF59-citrate, administered SC to anesthetized animals in 200 μl of inoculum. The recombinant Spike protein vaccine will be tested at the dose eliciting the highest neutralizing antibody titers in mice at day 35 after the second boost. The animals will be primed at day 0 and boosted at day 14 and 28. Peripheral blood will be collected at days 7, 21, and 35. Neutralizing and Spike-specific antibodies titers will be determined by inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively. Sampling Number Groups Treatment Dose/Route interval of ferrets 1 & 2 Rec-Spike protein Y μg or 2Y μg/SC 7, 21, 35 d 6 3 & 4 Rec-Spike protein + Y μg or 2Y μg/SC 7, 21, 35 d 6 MF59 5 Saline NA/SC 7, 21, 35 d 6

The 3 groups of ferrets, 6 animals per group, used for the immunogenicity studies above can then be used to assess efficacy of the recombinant Spike protein in protecting vaccinated animals from infection and/or disease. Anestethized animals will be challenged two weks after the last boost intratracheally with 10⁶ median tissue culture infectious dose unit (TCID₅₀) of the SARS-CoV Utah strain. Infection by SARS-CoV will be assessed by taking nasal, faringeal and rectal swabs from animals for 20 days after challenge as described (12). The presence of SARS-CoV in sample materials will be assessed by RT-PCR and infection assay of Vero cells. Animals will be monitored for clinical signs of SARS disease by assessing sleeping time, temperature, respiratory symptoms, diarrhea, body weight and survival. Protection will be determined by the magnitude and duration of virus shedding and by duration and severity of disease symptoms and percentages of surviving animals.

Example 10 Expression of Spike Protein for Vaccination

The SARS-CoV Spike glycoprotein was expressed in both full-length and truncated forms, using the nSh and nShATC pCMVIII constructs described above, both with hexahistidine tags. The vector constructs were evaluated for expression 48 hr after transfection into 293 cells and COS7 cells. The full-length Spike protein (nSh) was detected by western blot only in cell lysate, but not in culture media (FIG. 52).

The majority of SARS-CoV full-length Spike protein was expressed in transiently-transfected COS7 cells as a high molecular glycoprotein which ran at 540 kDa in non-reducing gels (FIG. 53). The gp540 is heat labile as indicated by the complete dissociation into monomeric forms (gp170 & gp180) by boiling, but it was resistant to DTT treatment. These data suggest that the recombinant Spike protein is noncovalently associated into a homotrimer (gp540). The presence of Spike protein in homotrimeric association also was confirmed in inactivated, purified SARS-CoV virion particles. Analysis of virion proteins by western blot under the same condition used for the characterization of recombinant Spike protein generated essentially identical results (FIG. 54).

Example 11 Spike Protein Processing

In order to characterize Spike protein processing, BHK-21 cells were infected with alphavirus replicon particles expressing the SARS-CoV full-length Spike. At 6 hoursr post-infection with an MOI of 5, infected cells were labeled for 1 hr with L-[³⁵S]methionine/cysteine and chased for up to 4 hours. The [³⁵S]-labeled spike protein was immunoprecipitated by anti-SARS rabbit serum and digested with Endo-H. Both digested and undigested proteins were analyzed by SDS-PAGE (4% polyacrylamide). As shown in FIG. 55, the full-length spike protein is synthesized as an Endo-H sensitive high-mannose glycoprotein (gp170, an ER form) that undergoes modification to an Endo-H resistant glycoprotein with complex oligosaccharides (gp180, a Golgi form). The conversion of gp170 into the gp180 form takes place within 2 hours (FIG. 56).

Example 12 High-Level Protein Expression

To develop a system for rapid expression of protein antigens, DNA transfection of 293 (human embryonic kidney) cells was used, to obtain milligram quantities of recombinant antigen. The most common method for culturing and transfecting 293 cells is in static or monolayer cultures. These procedures were modified by performing large-scale transfection of 293 cells in suspension and expanding the transfected cells in suspension culture for production of secreted or intracellular proteins. Several initial experiments were performed at the 100-milliliter scale cultures to determine optimum conditions, such as number of cells, type of transfecting reagent (FuGENE 6, Lipitoid or RO-1538) and the ratio of DNA to transfection reagent. Based upon pilot experiments, FuGENE 6 was the best transfecting reagent.

The kinetics of gene expression was compared to other viral envelope glycoproteins, and the data suggest that stable protein expression peaks around 72 to 96 hours post-transfection, depending upon the gene of interest, and then significantly decreases thereafter. Thus, using the optimum conditions, the transfection process was scaled from 100 ml to 4 liters. The 4 liter culture can be used for rapidly producing 2-10 milligrams of protein antigens. To facilitate antigen purification and also maximize the yield and recovery of the purified protein, transfection conditions were optimized by using serum-free medium.

Bulk transfection procedure has been used for the expression of truncated and full-length Spike antigens. The kinetics of expression for truncated form of the spike protein is presented in FIG. 56A. Expression of the truncated form of Spike protein peaked around 48 hrs and was stable until 72 hrs, therefore the cultures were harvested at 72 hrs post transfection.

Collected media were concentrated 20X and used for purification of truncated Spike protein by a very simple purification strategy where the truncated form of the spike was captured on GNA lectin followed by DEAE and ceramic hydroxyapatite column chromatography. The purified protein was analyzed on SDS-PAGE by silver stain (FIG. 56B) and also by western blot (FIG. 56C). Early efforts were able to purify the truncated form of the spike protein with >95% purity and approximately 50% recovery. The molecular mass of the truncated form of the Spike protein is approximately 170-180 kDa.

Full-length Spike protein was expressed in 293 cells using the bulk transfection strategy. The expression data suggest that, like the truncated form, expression peaked around 48 hrs post-transfection and remained stable until 72 hrs. However, contrary to the truncated form and as expected, full-length protein is not secreted, but rather is retained within the cells, as shown by the absence of any signal in western blots of cell culture supernatants. The full-length form of the protein was purified from Triton X-100 detergent-extracted cells. Full-length Spike protein was then captured on GNA lectin, followed by hydroxyapatite and SP chromatography. The calculated molecular mass of full-length spike protein is approximately 600 kDa, which is close to the theoretical mass for the trimer.

Example 13 SARS Virus Seed Cultures

A SARS-CoV reference seed virus propagated only in certified Vero cells will be used for the generation of the Master and Working Virus Seeds under GMP. A clinical specimen from the respiratory tract of a patient infected by the SARS-CoV is inoculated onto documented VERO cells, with certified culture media. Culture media containing the virus are harvested at 4 days post-infection and designated Passage 1 (P1). A second round of virus propagation is again performed in certified VERO cells with certified media, by inoculation of 1 ml per T-75 flask of 100 times diluted P1 virus. Culture supernatant was harvested at 3 days post-infection and stored at −80° C. as a P2 reference stock virus, without plaque purification.

Cell banks of Vero cells for further production of SARS-CoV are prepared from specific cell subsets that have not been used since the emergence of transmissible spongiform enephalopathies (e.g. since 1980). A research cell bank of these cells has been prepared using specified New Zealand-origin fetal bovine serum. From this research cell bank, a Master Cell Bank (MCB) is made under GMP conditions and using only specified and well-controlled media and supplements. The cell bank will is tested for absence of adventitious agents according to applicable US, EU, and international guidelines (see Points To Consider “Characterization of cell lines used to produce biologicals”, FDA/CBER 7/1993; ICH Q5D Draft 6 “Cell substrates”, Oct. 23, 1996; CPMP/ICH/294/95 “Note for Guidance on Quality of Biotechnological Products: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products (Step 4, 16. Jul. 1997); WHO final draft “Requirements for use of animal cells as in vitro substrates for the production of biologicals” Jul. 3, 1997). Tumorigenicity and identity testing is also required for this cell bank.

The reference virus is plaque-purified and expanded in certified Vero cells in the absence of FCS in order to generate Master and Working Seeds. Another option to help ensure purity and facilitate the assessment of safety of the Master Seed is to subject the SARS-CoV to pelleting and resuspension in PBS. The virus suspension is made up to 60% (w/w) sucrose with crystalline sucrose, transferred to a centrifuge tube and overlayed with 50, 40, 30, and 20% (w/w) sucrose solutions in PBS. The gradient is centrifuged for 72 h and then fractionated. The virus-containing fraction is diluted and the virions re-pelleted by ultracentrifugation. RNA from the virus pellet is isolated and transfected into certified Vero cells whereby the “infectious” positive-strand RNA will lead to the production of infectious virus, which can be plaque-purified and expanded to generate alternative Master and Working Seeds from purified virus RNA.

Viral seeds are tested for the absence of adventitious agents (see e.g. 21 CFR Revised as of Apr. 1, 1994, §630.35 Test for safety) and for identity, using a highly-specific neutralizing antiserum prepared from an independent source. Safety testing of viral seeds for vaccine purposes is done routinely by service laboratories. Broad-spectrum PCR testing can be used as an addition and/or alternative for testing.

Example 14 Scale-Up of Virus Production and Inactivation

A protocol for the production, inactivation, and purification of inactivated SARS-CoV with sufficient structural integrity to elicit protective neutralizing antibody responses in animal models involves: Vero cells are infected with virus at an M.O.I. of 0.01 in the absence of FCS and antibiotics; culture medium is collected, cleared by centrifugation, and inactivated with BPL, followed by confirmatory testing for complete inactivation; the inactivated material is filtered, subjected to MCS-column purification, and further purified by sucrose gradient centrifugation.

Several modifications and improvements can be developed when adapting this basic protocol to a larger scale for commercial use. Firstly, the cell culture and infection process can be adapted to roller bottles, as an intermediate step to allow rapid production for preliminary trials within existing BSL 3+ facilities. Full commercial production will typically use a fermentation process in a closed system, but a roller bottle system can be achieved more rapidly. The roller bottles do offer a true suspension culture system for Vero cells, which gives various technical and safety advantages over microcarrier cultures. Suspension cultures can be grown to any desired fermentation scale without interfering with the closed system between cell passages, as no trypsinization is required.

To scale up the infection process in roller bottles to 30-50 liters per batch, the optimum M.O.I. and harvesting periods for selected media and culture conditions should first be determined. For the larger scale, methods for harvesting and handling larger volumes of highly infectious material safely should be used, and so cell separation via centrifugation should be replaced by a method such as filtration through single-use filter cartridges.

The MCS-chromatography and the gradient purification steps described above can readily be scaled to a batch volume of up to 50 liters. For larger volumes, however, and for increased purity, ultrafiltration and sterile filtration steps will be used. Nuclease treatment to remove host cell DNA will also be included.

Example 15 Large Scale Analytical Methods

Analytical methods for the SARS coronavirus include virus titration methods, immunological and physico-chemical methods to quantitate and characterize the purified antigen (ELISA, PAGE, western blots using specific antisera against purified whole virus, etc.). Other analytical tests include: fast yield testing via asymmetric field flow separation and laser particle detection and counting; Western blot using specific antisera against individual viral proteins; and tests for residual host cell DNA.

Residual DNA testing is generally done by hybridization e.g. using a limit test. Such testing is performed according to methods already established and validated for other cell lines. As an alternative, the Threshold™ method may be used.

For producing specific antibodies, recombinant protein expression of all the ORFs from the structural and non-structural gene regions of the SARS-CoV is used. The ORFs can be cloned and expressed in E. coli and, if necessary, also in eukaryotic vectors such as baculovirus. This can provide sufficient amounts of purified soluble protein to immunize mice and rabbits to produce polyclonal and monoclonal antibodies against SARS proteins and to set up specific ELISA assays. Different expression vectors can be tested to maximize the yield of recombinant protein in a soluble form e.g. different vectors, one containing sequences coding for six N-terminal histidine residues and another containing a Glutathione-S-transferase protein fused to the C-terminus of the SARS protein. The recombinant proteins can be purified by single step column chromatography on either Nickel chelating Sepharose or Glutathione-Sepharose 4B resin. These procedures are very rapid and generally produce protein of 60-90% purity, which is suitable for raising specific antisera (Pizza et al. (2000) Science 287:1816-20). Five mice and two rabbits for each recombinant protein can be immunized SC with 20 and 50 μg recombinant protein, respectively, given in IFA as adjuvant, at day 0, 14 and 28. Sera are collected at day 7, 21 and 35 to assess specific titers before euthanasia of the animals for collection of blood and removal of spleens.

For the detection of impurities (e.g. Vero cell derived proteins) in the vaccine preparation, rabbit serum reactive against Vero-derived proteins can be used. Such antisera are obtained by immunizing rabbits with at least 10 μg of Vero cell lysate with CFA/IFA. The sera can be verified for reactivity against Vero-derived proteins in western blots. For more specific antisera against specific relevant cell-derived proteins that tend to be co-purified with the virus, mock-infected cell culture harvest that have undergone the purification process can be prepared and used for immunizing rabbits.

Methods to determine neutralization titers of sera from immunized animals and humans can be developed, without the constraints of using infectious SARS-CoV in a BSL-3+ laboratory. One such strategy will be to use recombinant antigens, particularly Spike protein or Spike-derived epitopes, and to develop ELISA assays for measuring antibodies against the target protein. Suitable epitopes allow a correlation to be established between the ELISA values and virus neutralization assay values. This approach provides a faster and more efficient (higher-throughput) comparison of specific and protective antibody titers. This ELISA test is also the ideal tool to monitor specific antibodies in safety trials, where several hundred animal sera must be tested.

Another strategy is to combine structural elements from both the pathogenic SARS-CoV and the non-pathogenic coronavirus mouse hepatitis virus (MHV) to construct chimeric virus-like particles (VLPs) that can be labeled. The assay is based on fusion between octadecyl rhodamine (R18)-labeled VLPs and cells (Hoekstra et al. (1984) Biochemistry 23:5675-81). The method relies on the relief of fluorescence self-quenching of R18 incorporated into VLPs upon fusion with cellular membranes. Coronavirus VLPs have been shown to mimic native virions with respect to their appearance in the electron microscope (EM) and their biological activities. As they do not contain viral RNA, however, then they cannot cause a productive infection (Vennema et al. (1996) EMBO J 15:2020-2028). The VLP system can be used for the mouse hepatitis virus (MHV) strain A59 (MHV-A59) )Godeke et al. (2000) J Virol 74:1566-15) containing a chimeric S protein. The protein chimera, consisting of the ectodomain of the SARS-CoV and the transmembrane and endodomain (64 C-terminal amino acid residues) from the MHV spike protein, can be co-expressed with the MHV M (membrane) and E (envelope) protein in OST-7 cells )Godeke et al.). VLPs secreted in the supernatant are harvested, purified and labeled with octadecyl rhodamine (R18) (Hoekstra et al). A constant amount of VLPs is incubated with a serial dilution of sera at 37° C. for 1 hour in a 96-well plate. Subsequently, cells expressing the receptor for the SARS-CoV, the angiotensin-converting enzyme 2 (ACE2) (Li et al. (2003) Nature 426:450-54) is be added and the extent of fusion can be measured with a fluorescence spectrophotometer.

A final strategy to monitor the ability of sera to inhibit cell-cell fusion interactions between cells expressing the SARS-CoV S protein and a human cell line expressing the angiotensin-converting enzyme 2 (ACE2), a functional receptor for SARS-CoV (Li et al.). This reporter gene-based assay uses the fluorescent shift (green to blue) of the fluorogenic substrate CCF2/AM (AM=acetoxymethyl) upon cleavage by β-lactamase (Bla) as read-out for cell-cell fusion (Zlokarnik et al. (1998) Science 279:84-88). For this assay, a BHK-derived cell line, stably expressing Bla and the SARS-CoV S protein is generated. In addition, a human cell line expressing ACE2 on its surface is used. BHK cells, expressing the S protein on their surface and Bla in their cytosol are incubated with serial dilutions of the sera to be tested for 1 h at 37° C. The cell line expressing the ACE2 is loaded with 1 μM CCF2/AM for 1 h at 22° C., washed twice with PBS, and co-cultivated with the BHK cells. In case of cell-cell fusion, Bla cleaves the substrate, resulting in a green blue shift with excitation at 409 nm. Inhibition of fusion by sera thus provides a detectable change.

Example 16 Stabilisation of Inactivated SARS-CoV

Although the purified inactivated SARS-CoV vaccine is capable of inducing potent neutralizing antibody responses in animals, it is relatively instable and can benefit from formulation to increase stability for an acceptable period of time. Suitable formulation changes include the use of various buffer systems, pH ranges, stabilizing excipients (e.g. sugars and sugar alcohols, amino acids, etc.) etc.. Stability testing can be conducted in real-time at normal storage temperatures, or can be conducted in an accelerated manner by using elevated temperatures. Vaccine stability can thus be increased to approximately one year or longer. Lyophilized vaccine formulation can also be used to extend shelf-life, possibly with further additives for stability during lyophilisation.

Example 17 Dose and Schedule Optimization for Inactivated Virus

Animal models of SARS-CoV infection have been reported, including mice, ferrets and macaques. As mentioned in example 4 above, mice immunized with the BPL-SARS-CoV vaccine achieve neutralizing antibody titers in the range of 1:100-1:1000, similar to levels found in convalescent patients, and are 100% protected from infection with a challenge virus. While the mouse challenge model is limited only to infection but not disease, ferrets and macaques are useful models of the human SARS disease. Two to four days after inoculation with SARS-CoV, both ferrets and macaques have been found to shed infectious SARS-CoV particles from the throat, nose and pharynx, as demonstrated by RT-PCR and/or virus isolation on Vero cells. At approximately the same time, the infected animals became lethargic, show respiratory distress and eventually die. Histologically, SARS-CoV infection in these animals associates with pulmonary lesions of different severity, similar to those found in biopsied lung tissue and autopsy material from SARS patients. With the availability of these models, preclinical studies with vaccines can be performed initially in mice for immunogenicity readouts, while efficacy of optimal doses and schedules can be assessed in the ferret and macaque models.

Initial studies in mice are used to determine the optimal dose and schedule required to elicit the highest levels of neutralizing antibody, with titers at least in the range of 1/100- 1/1000. In parallel to the assessment of neutralizing activity, other features of the humoral immune response and cellular immune responses can be investigated. In particular sera from immunized mice can be assessed for the isotype (IgG1 vs. IgG2a) of the Spike-specific antibody response. Also, the frequencies of splenic CD4+ T cells producing IFN-γ and IL-4 in response to BPL-SARS-CoV particles will be assessed by ELISPOT and ELISA. These experiments can provide insight into the quality of the T cell response helping the priming of a protective antibody response.

Increasing vaccine doses can be tested (e.g. from 5 to 20 μg of BPL-SARS-CoV alone or mixed with an equal volume of MF59-citrate), administered SC to anesthetized mice in 100 μl of inoculum. Groups of BALB/c mice, 10 per treatment, are immunized, with priming at day 0 and boosting at days 14 and 28. Secondary endpoints compare the kinetics of neutralizing vs. Spike-specific antibody titers and assess the Th1/Th2 profile of the specific immune response, and so neutralizing and Spike-specific antibody titers are assessed at days 7, 21, 35, and at 2, 3, 4, and 5 months after priming. The IgG2a and IgG1 titers of Spike-specific antibodies are determined at days 21, 35, and at 2, 3, 4, and 5 months after priming. Proliferation and IFN-γ and IL-4 production by splenic T cells against recombinant Spike protein from SARS-CoV are assessed at day 42, and at the end of the 5th month. Peripheral blood is collected at days 7, 21, 35, and at 2, 3, 4, and 5 months after priming. Spleen cells will be obtained at day 42 and at the end of the 5th month. Neutralizing and Spike-specific antibody titers and isotypes are determined by inhibition of infection of Vero cells and by ELISA, respectively. Proliferation of splenic cells is determined by ³[H]-thymidine uptake. Frequencies of splenic IFN-γ and IL-4 producing CD4⁺ T lymphocytes is determined by ELISPOT and FACS analysis.

Based on mouse results, the BPL-SARS-CoV vaccine can be tested in ferrets for the induction of protective neutralizing antibody titers. Ferrets are immunized according to a similar schedule as the mice and at the dose that elicits the highest neutralizing antibody titers in mice at day 35 after the second boost. Three groups of ferrets, 6 per treatment, are immunized with BPL-SARS-CoV, alone or mixed with an equal volume of MF59-citrate, administered SC to anesthetized animals in 200 μl of inoculum. The animals are primed at day 0 and boosted at days 14 and 28. Peripheral blood is collected at days 7, 21, and 35. Neutralizing and Spike-specific antibodies titers are determined by inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively. Each group of ferrets is used to assess efficacy of the BPL-SARS-CoV in protecting vaccinated animals from infection and/or disease. Anesthetized animals are challenged intratracheally, two weeks after the last boost, with 10⁶ median tissue culture infectious dose units (TCID₅₀) of the SARS-CoV CDC strain. Infection by SARS-CoV can be assessed by taking nasal, pharyngeal and rectal swabs from animals for 20 days after challenge (Martina et al. supra). The presence of SARS-CoV in sample materials can be assessed by RT-PCR and infection assay of Vero cells. Animals can be monitored for clinical signs of SARS disease by assessing sleeping time, temperature, respiratory symptoms, diarrhea, body weight and survival. Protection can be determined by the magnitude and duration of virus shedding, by duration and severity of disease symptoms, and by percentage of surviving animals. The formulation eliciting the highest neutralizing antibody titers at day 35 can then be tested against a two-fold higher dose of BPL-SARS-CoV given in the same formulation in the same regimen.

Additional studies can evaluate immunogenicity and efficacy of the candidate vaccine in non-human primates. Three groups of adult cynomolgus macaques, 4 per treatment, are immunized with BPL-SARS-CoV, alone or mixed with an equal volume of MF59-citrate, administered SC to anesthetized animals in 500 μl of inoculum. The BPL-SARS-CoV vaccine can be tested at the dose eliciting the highest neutralizing antibody titers in ferrets at day 35 after the second boost. The animals are primed at day 0 and boosted at 3 and 6 weeks. Peripheral blood is collected at weeks 1, 4, and 7. A secondary endpoint is to assess the Th1/Th2 profile of the specific immune response. Neutralizing and Spike-specific antibody titers and frequencies of peripheral blood CD4+ T cells producing IFN-γ and IL-4 in response to the recombinant SARS-CoV Spike protein is thus assessed at weeks 1, 4, and 7. Neutralizing and Spike-specific antibody titers can be determined by inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively. Intracellular cytokine staining and FACS analysis will be used to quantify IFN-γ- and IL-4-producing CD4⁺ T cells. The macaques can also be used to assess efficacy of the BPL-SARS-CoV in protecting vaccinated animals from infection and/or disease. Anesthetized macaques can be challenged two weeks after the last boost with 10⁶ median tissue culture infectious dose unit (TCID₅₀) of the SARS-CoV CDC strain in a 5 ml volume. A few drops of the virus can also be administered on each of the conjunctiva, 0.5 ml in the nose and the remainder in the trachea. Infection by SARS-CoV can be assessed by taking nasal, pharyngeal, and rectal swabs, and feces from animals for 20 days after challenge (Fouchier et al. (20030 Nature 423:240). The presence of SARS-CoV in sample materials can be assessed by RT-PCR and infection assay of Vero cells. Animals can also be monitored for clinical signs of SARS disease by assessing sleeping time, temperature, respiratory symptoms, diarrhea, body weight and survival. Protection can be determined by the magnitude and duration of virus shedding, by duration and severity of disease symptoms, and by percentage of surviving animals. Mice Group Treatment Dose/Route Sampling interval Number of mice 1-3 BPL-SARS-CoV 20, 10, 5 μg/SC 7, 21, 35 d; 10 per dose level 2, 3, 4, 5 m; 4-6 BPL-SARS-CoV 20, 10, 5 μg/SC 42 d 10 per dose level 7-9 BPL-SARS-CoV MF59 20, 10, 5 μg/SC 7, 21, 35 d; 10 per dose level 2, 3, 4, 5 m; 10-12 BPL-SARS-CoV MF59 20, 10, 5 μg/SC 42 d 10 per dose level 13 MF59 NA/SC 7, 21, 35 d; 10 + 10 (sacrificed at 2, 3, 4, 5 m; 42 d and end 5 m) 14 Saline NA/SC 7, 21, 35 d; 10 + 10 (sacrificed at 2, 3, 4, 5 m; 42 d and end 5 m)

Ferrets Sampling Group Treatment Route interval No. of ferrets 1 BPL-SARS-CoV SC 7, 21, 35 d 6 2 BPL-SARS-CoV-MF59 SC 7, 21, 35 d 6 3 Saline SC 7, 21, 35 d 6

Macaques No. of Group Treatment Route Sampling interval macaques 1 BPL-SARS-CoV SC 1, 4, 7 w 4 2 BPL-SARS-CoV - SC 1, 4, 7 w 4 MF59 3 Saline SC 1, 4, 7 w 4

Example 18 Human T Cell Responses

Asia prelude to initiation of clinical studies in humans, the reactivity of peripheral blood T lymphocytes from healthy donors with different HLA haplotypes can be assessed using the in vitro priming technique (Abrignani et al. (1990) Proc Natl Acad Sci USA 87:6136-40). The aim of this study is to have a first indication of the immune-dominant T cell epitopes in SARS-CoV proteins. Briefly PBMCs from 20 healthy donors with different HLA haplotypes will be cultured in medium containing 5% autologous serum, in the presence of different concentration of SARS-BPL-CoV particles in the range from 0.5 to 20 μg/ml. The expression of activation markers will be assessed after 24 and 48 hours. Frequencies of IFN-γ- and IL-4-producing T lymphocytes will be assessed after 12 h and after 15 days in culture, in the presence of 100 U/ml recombinant human IL-2. Activated and cytokines producing CD4 T lymphocytes will be sorted and eventually cloned as single cells using FACS technologies. The CD4+ T cell repertoire from human subjects with different HLA will be assessed by proliferation assays of the CD4+ T cell lines and clones against autologous EBV-transformed cell lines loaded with 15-mer overlapping peptides from the most relevant structural and non structural protein of the SARS-CoV.

When moving to actual human trials, safety and immune responses will be evaluated in healthy adults following intramuscular immunization with escalating doses of the BPL-inactivated SARS-CoV vaccine, with MF59 adjuvant being included or omitted depending on preclinical data. Three/four immunizations will be given at 0, 1, 6 months in the first cohort, and at 0, 1, 2, 6 months and 0, 2, 6 weeks in the second and third cohorts respectively. The trial will be observer blind and placebo controlled. Subjects will be randomized into each dose level. Immune response parameters to be measured will include serum neutralizing antibodies, ELISA antibodies and peripheral blood IFN-gamma-producing CD4+ T cells by intracellular cytokine staining. Antigen Administration No. treated No. subjects Sampling Group dose (μg) schedule subjects with placebo interval A1 10 0, 1, 6 months 18 6 0, 1, 2, 6, 7 mos A2 20 0, 1, 6 months 18 6 0, 1, 2, 6, 7 mos B1 10 0, 1, 2, 6 months 18 12 0, 1, 2, 6, 7 mos B2 20 0, 1, 2, 6 months 18 12 0, 1, 2, 6, 7 mos C1 10 0, 2, 6 weeks 18 12 0, 2, 6, 10, 30 wks C2 20 0, 2, 6 weeks 18 12 0, 2, 6, 10, 30 wks

Example 19 Selection of CHO Cell Lines for Spike Protein Expression

Methods for the derivation of Chinese Hamster Ovary (CHO) cell lines that stably express viral envelope glycoproteins that are conformationally intact, appropriately glycosylated and efficiently bind neutralizing antibodies are well established for HIV and HCV (Srivastava et al. (2002) J Virol 76:2835-47; Srivastava et al. (2003) J Virol 77:11244-259; Heile et al. (2000) J Virol 74:6885-92). The same techniques can be applied to SARS-CoV, to generate two different stable CHOK-1 cell lines producing either full-length or truncated SARS Spike proteins. The Spike proteins can be expressed using the constructs described herein, but without the hexa-His tags. These proteins can compared for their ability to produce neutralizing antibodies in immunized animals as well as for their expression levels in CHOK-1 cells.

A pCMV3 vector expressing Spike can be used for the derivation of stable CHOK-1 cell lines, containing the CMV enhancer/promoter, ampicillin resistance, and a fused DHFR and attenuated neomycin gene for selection purposes. Stable cell lines can produced using the neomycin selection system in CHOK-1 cells. Clones can be sequenced to verify the integrity of the insert, and transient transfections can be performed using Trans-LT1 polyamine transfection reagent (PanVera Corp., Madison, Wis.) to assess the expression level and also the integrity of the expressed protein by ELISA and western blot analysis.

Initial CHO cells will be selected to be free from TSE/BSE contaminants and risks according to relevant regulatory standards. To construct cell lines, procedures involve transfection, primary screening with selective medium, followed by subcloning to assure purity of cell lines. Cell supernatants can be assayed using an antigen capture ELISA to quantify expression levels at all stages of selection and amplification. For full-length Spike expression, methanol fixed cells can be screened for internal expression by immunofluorescent staining using a rabbit anti-SARS antibody. Successive measurements at the T75-flask stage of expansion canbe employed to assure stability of expression levels. The molecular mass and integrity of the expressed proteins can be checked by PAGE both under native and reducing and denaturing conditions, followed by immunoprobing.

The pCMV3 vectors expressing SARS-CoV Spike proteins in either full-length or truncated forms can be introduced into CHOK-1 cells using the Trans-LT-1 reagent and non-selective media. 24-48 hours post-transfection, depending on cell density, cells are split at a 1:5 ratio and the medium can be changed to selective media containing neomycin at 500 μg/ml. Any bovine serum used in these procedures will be from TSE-free sources that meet regulatory standards. Ten to fourteen days later, individual colonies can be picked and transferred to 96 well plates and cultured in complete non-selective medium. When approximately 80% of the wells are confluent, 24 hour supernatants can be screened by Spike capture ELISA. For initial expression of full length Spike protein, cells can be fixed with methanol and screened by immunofluorescent staining using a rabbit anti-SARS antibody. After low-expressing cell lines have been eliminated and there are fewer than 20-30 cell lines, capture ELISA and western blots can then be used to determine the expression level after cell lysis. A portion of each cell line can be pelleted, weighed and lysed in 1% Triton lysis buffer for determination of expression levels. Three to four clones producing the highest levels of spike protein in correct structure and conformation can be expanded to three-liter bioreactors and adapted to low serum suspension culture conditions for scale-up.

The antigen capture ELISA assay for the SARS spike protein can be performed using 96 well flat-bottom plates coated with 250 ng per well of purified immunoglobulin obtained from rabbit sera that were immunized with inactivated SARS virus. Supernatant or lysate samples are added and incubated for 2 hours at 37° C. Bound antigen is reacted against pooled SARS^(+ve) serum or high affinity monoclonal antibody either human or mouse against SARS spike protein and detected using appropriate species-specific peroxidase-conjugated second antibody. The plates are developed using TMB substrate (Pierce, Rockford, Ill.), read at a wavelength of 450 nm, and the concentration of protein per ml sample is derived from a standard curve (OD vs. protein concentration) based on serial dilutions of a known concentration of recombinant spike protein.

The immunoprobing analysis will also be performed following the standard methods described by Srivastava et al. (2002) supra. Briefly, 10-20 μl of the sample is analyzed on 4-20% SDS PAGE under non-reducing/denaturing conditions with mild heating. The proteins are then transferred onto nitrocellulose membranes and reacted against polyclonal anti-Spike rabbit serum, followed by anti-rabbit Ig conjugated to Alexa 688 (Molecular Probes, Oreg.). The blots are scanned using an infrared imaging system.

The highest expressing candidate cell lines will be screened for Spike protein expression and stability in small-scale (3 liter) perfusion bioreactors. The candidate clones will be further evaluated for level of expression as well as integrity of expressed protein, and subsequently tested for expression stability in the absence of selection. The selected clones also will be tested for maintenance of the DNA sequence integrity of the integrated SARS spike protein gene. To quickly monitor the expression levels in small flasks and in the three liter evaluation cultures, a lectin-based process (Gluvanthus Nivalis lectin) has been developed to isolate SARS spike protein to a degree of purity that allows semi-quantitation and characterization of the protein in CHO supernatant. Full-length Spike protein will be obtained from Triton X-100 detergent extracted cells and then captured on GNA lectin, followed by hydroxyapatite and SP chromatograph. Eluted protein is then characterized by: (1) polyacrylamide gel electrophoresis (PAGE) and Coomassie staining, (2) immunoprobing with anti-SARS rabbit sera, (3) structural characterization using size exclusion chromatography (SEC), as well as mass spec analysis using MALDI-TOF.

Productivity from the CHO cell line expressing SARS spike protein should be at least 2 mg/L and for full-length Spike protein will be 3 mg/100 gm of cells, at steady-state cell density. Yield from one 45 day, 2.5-liter bioreactor will be ˜1000 mg crude protein.

Example 20 Purification of Spike Protein for Human Vaccines

To purify SARS spike protein for the purpose of producing GMP grade material for human use, the following basic process is used, with all steps being performed at 2-8° C.: the starting material, concentrated CHO cell culture supernatant (20-30×) is thawed and filtered through a 0.45 μm membrane; this material is heavily contaminated proteins from culture, as well as DNA; the first purification step is affinity chromatography using Gluvanthus Nivalis (GNA), a lectin that preferentially recognizes terminal mannose containing carbohydrates; glycosylated proteins, including SARS spike protein are captured and non-glycosylated proteins, as well as DNA, do not bind to this column; the GNA column is followed by two chromatographic steps operated in the flow through mode; the anion exchanger, DEAE, and ceramic hydroxyapatite (cHAP); DEAE binds some contaminating supernatant proteins and DNA, whereas cHAP binds any contaminating serum proteins; full-length Spike protein is purified from the cell pellet; the cells are lysed with Triton X-100 and full-length Spike protein is then captured on GNA lectin, followed by hydroxyapatite and SP chromatography.

The purified SARS spike can be further treated to remove adventitious viruses: viral inactivation at pH 3.5 for 1 hour; the sample is then concentrated and diafiltered into a buffer at pH 4 and finally captured the purified protein using SP resin; the spike protein binds to this resin and many viruses flow through.

The spike protein is eluted, concentrated and diafiltered into formulation buffer. This formulated bulk product is then filtered through a DV50 viral removal membrane followed by filtration through a 0.2 μm membrane. The formulated bulk is filled into suitable containers e.g. into 3.0 ml vials, in a class 100 laminar flow hood.

In process testing at each step of the purification includes protein concentration, endotoxin (LAL), bioburden, and recovery.

Prior to human administration, a test for potency will evaluate the specific ability of the vaccine in an in vitro or in vivo test to effect a given response. The in vivo immunogenicity will be determined by dosing groups of 10 mice with various doses of the protein antigen. Sera will be analyzed for the presence of IgG antibodies using an ELISA. The criterion for passing will be based upon the number of vaccine treated animals that are seropositive compared to a reference standard. Other tests include General Safety, sterility, purity, identity of the vaccine (using an ELISA specific for Spike protein), and quantity & protein concentration (UV spectrophotometric absorbance procedure based on the molar absorbance of the aromatic amino acids).

Stability testing will be performed on the bulk drug substance and on the final container product. Bulk product will be evaluated at temperatures of −60° C. (recommended storage condition), 25±2° C. and 40±2° C. protected from light, at time points of 0, 3, 6, 9, 12 months. Final container product will be tested at temperatures of −60° C., and inverted at 5±3° C., 25±2° C., and 40±2° C. at time points of 0, 3, 6, 9, 12 months. Stability-indicating assays may include appearance, pH, protein content, SDS-PAGE, size exclusion HPLC, and container/closure integrity, performed on single samples of bulk and triplicate vials of final container material.

The protein purified in this way can be evaluated in mice, rabbits and ferrets as described in, and based on the results of, examples 4, 5, 8 and 9 above.

Initial experiments will be performed in mice to determine optimal dose and schedule of the GMP Spike protein required to elicit the highest levels of neutralizing antibody, with titers at least in the range of 1/100- 1/1000. Spike protein will be tested in the range from 5 to 40 μg, alone or mixed with an equal volume of MF59-citrate, to anesthetized mice in 100 μl of inoculum. Groups of BALB/c mice, 10 per treatment, will be immunized. The animals will be primed at day 0 and boosted at days 14 and 28. Secondary endpoints will be to compare the kinetics of neutralizing vs. Spike-specific antibody titers and to assess the Th1/Th2 profile of the specific immune response. Neutralizing and Spike-specific antibody titers will be assessed at days 7, 21, and 35 and at 2, 3, 4, and 5 months after priming; the IgG2a and IgG1 titers of Spike-specific antibodies will be determined at days 21 and 35, and at 2, 3, 4, and 5 months after priming; proliferation and IFN-γ and IL-4 production by splenic T cell against the recombinant Spike protein from SARS-CoV will be assessed at day 42 and at the end of the 5th month. Peripheral blood will be collected at days 7, 21, and 35 and at 2, 3, 4, and 5 months after priming; spleen cells at day 42 and at the end of the 5th month. Neutralizing and Spike-specific antibody titers and isotypes will be determined by inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively. Proliferation of splenic cells will be determined by ³[H]-thymidine uptake. Frequencies of splenic IFN-γ and IL-4 producing CD4+ T lymphocytes, will be determined by ELISPOT and FACS analysis.

Next, the optimal dosing and schedule for recombinant Spike vaccine will be determined in ferrets. Based on the mouse results, the Spike vaccine eliciting the highest antibody neutralizing titers will be tested against a two-fold higher dose of recombinant Spike protein given in the same formulation. Three groups of ferrets, 6 per treatment, will be immunized SC under anesthesia with 200 μl of inoculum. The animals will be primed at day 0 and boosted at days 14 and 28. Peripheral blood will be collected at days 7, 21, and 35. Neutralizing and Spike-specific antibodies titers will be determined by inhibition of SARS-CoV infection of Vero cells and by ELISA, respectively. Similar to the previous ferret studies, each group of animals will be used to assess efficacy of the vaccine in protecting immunized animals from infection and/or disease.

Immunogenicity and efficacy of the candidate vaccine also will be evaluated in nonhuman primates. Three groups of adult cynomolgus macaques, 4 per treatment, will be immunized with recombinant SARS-CoV Spike protein, alone or mixed with an equal volume of MF59-citrate, administered SC to anesthetized animals in 500 μl of inoculum. The Spike protein vaccine will be tested at the dose eliciting the highest neutralizing antibody titers in ferrets at day 35. The animals will be primed at day 0 and boosted at 3 and 6 weeks. Peripheral blood will be collected at weeks 1, 4, and 7. A secondary endpoint will be to assess the Th1/Th2 profile of the specific immune response, as described above (neutralizing and Spike-specific antibody titers, frequencies of peripheral blood CD4+ T cells producing IFN-γ and IL-4 in response to the recombinant Spike protein, assessed at at weeks 1, 4, and 7).

Finally, human phase I, placebo-controlled, dose-escalation, safety/immunogenicity trials will be performed for the IM recombinant SARS vaccine with MF59 adjuvant. The trial will evaluate safety and immune responses in healthy adults following immunization with escalating doses of SARS recombinant vaccine with MF59 adjuvant, administered intramuscularly. Three/four immunizations will be given at 0, 1, 6 months. The trial will be observer blind and placebo controlled. Subjects will be randomized into each dose level. Immune response parameters to be measured include serum neutralizing antibodies, ELISA antibodies and peripheral blood IFN-γ-producing CD4+ T cells by intracellular cytokine staining: Vaccine No. of Antigen No. of subjects dose Administration treated with placebo Sampling Group (μg) schedule subjects (MF59) interval A1 50 0, 1, 6 months 18 6 0, 1, 2, 6, 7 months A2 100 0, 1, 6 months 18 6 0, 1, 2, 6, 7 months

Example 21 Comparison of Inactivated Virus and Purified Spike Protein

Immunogenicity and efficacy of the inactivated virus vaccine and the purified Spike protein can be compared in non-human primates. Three groups of adult cynomolgus macaques, 4 for treatment, will be immunized with recombinant SARS-CoV Spike protein from CHO cell lines or with BPL-SARS-COV, given in the dose and formulation eliciting the highest neutralizing antibody titers in previous immunogenicity challenge experiments, administered SC to anesthetized animals in 500 μl of inoculum. The animals will be primed at day 0 and boosted at 3 and 6 weeks. Peripheral blood will be collected at weeks 1, 4, 7. A secondary endpoint will be to assess the Th1/Th2 profile of the specific immune response, as described above. Sampling No. of Group Treatment Dose/Route interval macaques 1 Rec-Spike protein + or − Y μg/SC 1, 4, 7 w 4 MF59 2 BPL-SARS-CoV + or − Y μg/SC 1, 4, 7 w 4 MF59 3 Saline NA/SC 1, 4, 7 w 4

Example 22 Expression in Yeast

Yeast is a useful and inexpensive eukaryotic expression system. Yeast-expressed proteins are used in recombinant hepatitis B virus vaccines, and recombinant SARS antigens may also be expressed in yeast for vaccine purposes. Yeast-expression is also convenient for the production of antigens for preparing monoclonal and polyclonal antitobodies, or for use in serological assays.

The nucleocapsid protein (N) and two different versions of the spike glycoprotein (S) from SARS coronavirus FRA strain (AY310120) were cloned for expression in S. cerevisiae:

SARS N: aa 1-422 (coordinates 28120-29388 of AY310120 strain)—FIG. 65

SARS spike: aa 14-1195 (transmembrane domain and cytoplasmic tail deleted)—FIG. 66

SARS spike: aa 14-662 (S1 domain)

To make the S1 construct, a XhoI-NotI fragment of approximately 3733 bp encoding the full-length spike glycoprotein was the starting point. PCR was used to amplify the full-length gene in two pieces: XbaI-BlnI of 2440 bp and BlnI-SalI of 1306 bp. These fragments were subcloned into commercial vectors (Novagen): pT7Blue2 XbaI-BlnI (5′ end of spike glycoprotein) and pT7Blue2 BinI-SalI (3′ end of spike glycoprotein; FIG. 58), respectively. The following primers were used in the subsequent PCR reactions: Spk-1 (5′) SEQ ID NO: 9785; Spk-2 (5′) SEQ ID NO: 9786; Spk-3 (5′) SEQ ID NO: 9787; Spk-4 (5′) SEQ ID NO: 9788.

E. coli HBO101 competent cells were transformed with the PCR ligation product and plated on Luria agar plates, containing 100 μg/ml ampicillin. The desired clones were identified using miniscreen DNA analysis. After sequence verification and plasmid amplification of the desired subclones, it was desirable to eliminate the internal SalI site present in the XbaI-BlnI portion of the spike sequence in order to facilitate future cloning into the yeast expression vector (BamHI-SalI). Therefore, we prepared a CelII-MfeI vector from the pT7Blue2 XbaI-BlnI (5′ end Spike) subclone to eliminated a 143 bp sequence containing the SalI site. Kinased oligos DSI-6 (SEQ ID NOS: 9789-9794) were then ligated into the CelII-MfeI vector to replace the 143 bp that were removed to mutate the SalI site (no aa changes), creating pT7Blue2.XbaI-BlnIΔsal.

The 5′ XbaI-BlnI (from pT7Blue2.XbaI-BlnI ΔSal) and the 3′ BlnI-SalI (from pT7Blue2 BlnI-SalI) spike glycoprotein inserts were gel-purified and ligated them into the p893-1 XbaI-SalI vector (a vector derived from pLitmus 38 (New England Biolabs) with the alpha-factor leader sequence cloned into the BamHI-SalI sites of the MCS). The resulting full-length SARS Spike coding sequence was named p893-1.SARS Spike 1255 #9 (FIG. 58).

E. coli HB 101 competent cells were transformed with the oligo replacement ligation product and plated on Luria agar plates, containing 100 μg/ml ampicillin. The desired clones were identified using miniscreen DNA analysis. After sequence verification of the positive clones, pT7Blue2 Xba-Bln ΔSal was chosen for use as a template for PCR reactions to amplify the Spike S1 1967 bp Xba-Sal fragment. The fragment was then subcloned into the p893-1 Xba-Sal vector, sequence verified, and named it p893-1.Spike S1 #11 (FIG. 59).

In order to clone into the S.cerevisiae expression vector, pBS24.1, the 5′ end of the S1 sequence had to be modified from XbaI to HindIII to allow ligation with the 3′ HindIII end of the ADH2/GAPDH BamHI-HindIII promoter fragment. From pT7Blue2 Xba-BlnΔSal (described above) an AgeI-SaII 1943 bp fragment was gel-purified. This fragment was ligated along with a synthetic pair of HindIII-AgeI 30 bp kinased oligos (S1-1+S1-2 creating the necessary 5′ HindIII site) into the pSP72 HindIII-SalI commercial subcloning vector (named pSP72.SARS Spike S1 #2; FIG. 59). S1-1 had SEQ ID NO: 9795 and S1-2 has SEQ ID NO: 9796.

After sequence verification of the positive clone from miniscreen DNA analysis, the HindIII-SalI fragment was gel purified. The 1365 bp BamHI-HindIII ADH2/GAPDH promoter fragment was ligated along with the 1973 bp HindIII-SalI S1 fragment into the pBS24.1 BamHI-SalI vector creating the genetically engineered pd.SARS Spike S1 #2 expression plasmid (FIG. 60).

S. cerevisiae strain AD3 was transformed with pd.SARS Spike S1 #2 and single transformants were checked for expression after depletion of glucose in the medium. The recombinant protein was expressed at high levels in yeast, as detected by Coomassie blue staining. In particular, yeast cells were transformed with the SARS S1 expression plasmid using the Invitrogen S.c. EasyComp™ Transformation Kit. Expression in shown in FIG. 57.

To express Spike 1195 protein, which does not contain the trans-membrane (TM) region or cytoplasmic tail that are present in the full-length SARS construct, the following series of genetic manipulations was performed:

From pT7Blue2 BlnI-SalI #11 (described above) a BlnI-Dral 1056bp fragment was gel purified. This fragment was ligated with a synthetic pair of 68 bp DraI-SalI kinased oligos (DRS1+2; SEQ ID NOS: 9797 & 9798) into a pT7Blue2 BlnI-SalI vector (FIG. 61). E. coli HB101 competent cells were transformed with the oligo replacement ligation product and plated on Luria agar plates, containing 100 μg/ml ampicillin. The desired clones were identified using miniscreen DNA analysis. After sequence confirmation the clone was named pT7Blue2 BInI-Sal Spike 1195 #7. The 1126 bp BlnI-SalI fragment encoding the 3′ end of the Spike 1195 was gel purified (FIG. 61).

In order to generate the XbaI-SalI Spike 1195 fragment, the 3109bp XbaI-PciI fragment was isolated from the p893-1.SARS Spike 1255 #9 (described above) and a 457bp PciI-SalI fragment from pT7Blue2.SARS Spike 1195 #7 (described above). The two fragments were cloned into the p893-1 XbaI-SalI vector, creating the p893-1.SARS Spike 1195 #34 plasmid (FIG. 62).

To clone SARS Spike 1195 into the pBS24.1 Saccharomyces cerevisisae expression vector, it was necessary to modify the 5′ end of the SARS Spike 1195 from XbaI to HindIII, as done for the Spike S1 expression clone described above. To begin, the 2416 bp AgeI-BlnI fragment was isolated from p893-1.SARS Spike 1195 #34. This fragment was ligated with the synthetic HindIII-AgeI 30 bp oligos (described above to generate the S1 protein for expression in S. cerevisiae) into the pT7Blue2 HindIII-BlnI vector. E. coli HB101 competent cells were transformed with the oligo replacement ligation product and plated on Luria agar plates, containing 100 μg/ml ampicillin. The desired clones were identified using miniscreen DNA analysis. After sequence verification of the positive clone and plasmid amplification of pT7Blue2.SARS 1195 5′ HindIII-BlnI #10 (FIG. 63), we isolated a 402 bp HindIII-NcoI fragment and the 2044 bp NcoI-BlnI fragment (FIG. 63). It was necessary for the HindIII-BlnI isolation to be done in two steps to avoid cloning issues related to the internal HindIII site located at nucleotide number 1319 of the spike 1195 protein.

To assemble the BamHI-SalI expression cassette of Spike 1195 into the pBS24.1 vector E.coli HB101 competent cells were transformed with the the BamHI-HindIII (ADH2/GAPDH promoter), HindIII-NcoI 402bp fragment, NcoI-BlnI 2044 bp and the BInI-Sall 1126 bp fragments into the pBS24.1 BamHI-SalI vector. The samples were plated on Luria agar plates, containing 100 μg/ml ampicillin. The desired clone was identified using miniscreen DNA analysis, thus creating the genetically engineered pd.SARS Spike 1195 #10 (FIG. 64).

S.cerevisiae strain AD3 was transformed with pd.SARS Spike 1195 #10 and single transformants were checked for expression after depletion of glucose in the medium. The recombinant protein was detected by Coomassie blue staining. In particular, yeast cells were transformed with the SARS 1195 expression plasmid using the Invitrogen S.c. EasyComp™ Transformation Kit.

Example 23 Expression in Mammalian Cell Lines

cDNA fragments containing the S protein ORF of 1255 amino acids were amplified by RT-PCR from SARS viral RNA (Frankfurt isolate) grown in Vero cells. The amplified PCR fragments were cloned into pBlueScript vector, sequenced, and consensus spike sequence was assembled to create a full-length SARS spike clone, pBSnSh. In vitro transcription of pBSnSh followed by translation in a rabbit reticulocyte lysate resulted in the production of single polypeptide with an estimated molecular mass of ˜140 kDa.

The insert of this plasmid was recloned via XhoI and Not I into a mammlian expression vector pCMVIII (Srivastava et al. (2003) J. Virol. 77:11244-11259) to create a construct, nSh (FIG. 74A). A PCR fragment containing a spike protein of 1195 amino acid, which was deleted for transmembrane (TM) domain and cystein-rich cytoplasmic tail (Cy) was amplified and cloned pCMVIII vector to generate the contstruct nShATC (FIG. 74B). Both constructs were tagged with six histidine residues at the C-terminus in order to aid in their characterization. The Xho I/Not I fragment without a histidine tag also was subcloned into the alphavirus replicon vector backbone pVCRchim2.1 for use in the production of an alphavirus replicon particle chimera that expresses S protein. Production and characterization of the replication defective alphavirus vector particles was performed essentially as described previously (Perri et al. (2003) J. Virol. 77:10394-10403; Polo et al. (1999) PNAS USA. 96:4598-4603). The resultant alphavirus vector particles were named as VEE/SIN.

COS7 cells and BHK-21 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37° C. and 5% CO₂ in air. COS7 cells were transfected with expression plasmids (nSh, nShΔTC) using a transfection kit (TransIt-COS, Mirus) following the manufacturer's protocol. The cells were washed once with ice-cold PBS and lysed with 1× Lysis buffer (20 mM MOPS, 10 mM NaCl, 1.5 mM MgCl₂, and 1% Triton X-100) containing complete mini protease inhibitor (Roche). After a 30-min incubation on ice, the debris was cleared by centrifugation. The cleared lysate was either purified or used directly in western blotting.

To purify secreted spike proteins, medium from transfected cells was collected and subjected to centrifugation at 12,000 rpm for 10 min to remove cellular debris. The cleared medium was applied to a ConA-agarose column (Vector Lab). The column was washed extensively with 20 mM sodium phosphate buffer, and then the bound proteins were eluted with 1M methyl α-D-mannopyranoside (MMP), 1M NaCl in 20 mM sodium phosphate buffer. Column fractions containing SARS-CoV spike proteins were applied to MagneHis Protein purification system (Promega) following the protocol suggested by the manufacturer.

For western blot analysis, proteins were separated by 4-20% SDS-PAGE and then transferred electrophoretically to nitrocellulose membrane (Invitrogen). Membrane was blocked in blocking buffer (5% skim milk and 0.1% Tween 20 in PBS) and incubated with indicated antibody at room temperature for 1 hr, washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibody (Biosource) followed by chemiluminescence (ECL system, Amersham) and exposed by X-ray films. The antibodies used were a mouse monoclonal anti-histidine antibody (anti-His•tag Mab, Novagen), a rabbit polyclonlal antipeptide antibody against SARS-CoV spike proten (SmPab, Abgent), or rabbit anti-SARS sera (2BE) obtained by immunization of rabbits with purified SARS-CoV virion. The latter has a cell culture neutralizing titer of 1/2,500. Unless stated otherwise, antibody was used at 1/1,000 for anti-histidine antibody and SmPab and 1/10,000 for anti-SARS rabbit sera.

Some spike proteins were treated with Peptide-N glycosidase F (PNGase F). Cell lysates were diluted in 0.5% SDS and 1% β-mercaptoethanol and denatured at 100° C. for 10 min. After 2-fold dilution with 1% NP-40 in 50 mM sodium phosphate (pH 7.5), the samples were treated with PNGase F (NEB) at 37° C. for 1 hr. Enzyme-treated samples were analyzed by 4-12% SDS-PAGE in reducing condition. For a partial digestion with the PNGase, the cell lysates were diluted with 50 mM sodium phosphate (pH 6.0) containing 0.75% Triton-X and treated with PNGase F (Calbiochem) at 37° C. for 3 hr. Enzyme-treated samples were analyzed by 4-20% SDS-PAGE in nonreducing condition.

Western blots of cells 48-hours after transfection are shown in FIG. 75. The S protein was detected in cell lysates as a doublet with estimated molecular weight of ˜170-180 kDa, when the lysate was boiled and analysed under reducing SDS-PAGE conditions (FIG. 75A, lane 3). This doublet appears to result from differential glycosylation of one polypeptide product since pre-treatment of the cell lysate with PNGase F reduced the doublet to a single species of ˜140 kDa (FIG. 75A, lane 4). This is the expected size predicted from the aa sequence for a full-length, intact polypeptide product. This experiment indicates that the full length SARS-CoV S is expressed in mammalian cells as a single, uncleaved polypeptide, but in two differentially glycosylated forms, gp170 and gp180 respectively. Unlike the two S glycoforms encoded by the full-length sequence, none of which were secreted, the SΔ protein product was detected both in cell lysates (FIG. 75A, lane 5) as well as in the cell culture medium (FIG. 75B, lane3) as a single species of 160 kDa.

In order to further characterize the intracellular processing of the S protein, and as described above, BHK21 cells were infected with defective alphavirus particles expressing the full-length S. At 6 hr post infection with a MOI of 5, infected cells were pulse labeled for 1 hr with L-[³⁵S] methionine/cysteine and chased for 2 or 4 hours. The [³⁵S]-labeled S protein was immuno-precipitated using the rabbit antiserum raised against inactivated, purified virus and then digested with Endo H. The Endo H treatment involved dilution with a sample buffer (50 mM sodium phosphate, 0.1% SDS, 50 mM DTT, pH 6.0) and boiling for 5 min. After denaturation, the samples were further diluted with 0.75% Triton-X 100 and treated with endoglycosidase H (Endo H) following manufacturer's protocol (Calbiochem) for 3 hr at 37° C. Enzyme-treated samples were added with gel loading buffer containing 0.1% SDS and DTT and analyzed by 8% SDS-PAGE.

Both digested and undigested proteins were boiled in SDS and analysed by reducing SDS-PAGE (FIG. 55). After a 1-hr pulse, the S protein was apparent as a single gp170 component that was Endo H sensitive (lanes 1 and 2). After a 2-hr chase, a new species (gp180) was present along with gp170 in approximately equal proportions (lane3). After a 4-hr chase, the gp180 species was the dominant S protein component (lane 5) that was now Endo H resistant (lanes 5 and 6). This data is consistent with gp170 being an ER-resident glycoprotein containing high mannose chains and with gp180 corresponding with a Golgi-processed glycoprotein containing Endo H-resistant complex oligosaccharides.

The Endo H sensitivity of the C-terminus deleted SΔ protein purified from cell culture media ws also tested. As shown in FIG. 76, the SΔ observed within cell lysates was found to be Endo H sensitive (lanes 1 and 2), while the secreted SΔ in cell culture media was Endo H resistant (lanes 3 and 4). This result is consistent with this glycoprotein being synthesized in an immature form in the ER prior to transfer to the Golgi where the complex carbohydrate is added and the protein then secreted.

As already described, the S protein expressed in COS7 cells was detected as a gp170/gp180 doublet in western blot analyses of cell lysates that were fully denatured by boiling in the presence of DTT. However, the majority of S protein was detected as a high molecular glycoprotein in the 440-669 kDa range when the same cell lysate was not heat-denatured prior to western blot analysis using SDS-PAGE (FIG. 77, lane 1). The ˜500 kDa species was resistant to 10 mM DTT treatment (lane 3) and not dissociated into the monomeric form unless the lysate was first heat-denatured at 100° C. (lane 4). In contrast, oligomeric form of a test protein (Thyroglobulin) of which quaternary structure is held by disulfide-linkage was converted into subunit form by the 10 mM DTT treatment. These data suggest that the ˜500 kDa oligomeric form of S protein is not disulfide-linked and is heat labile. To confirm the heat-sensitivity of the ˜500 kDa species of S protein, the heat-denaturation experiment was repeated but without DTT. As shown in FIG. 78, heat denaturation of 500 kDa protein at 100° C. alone was sufficient to convert it into gp170/180 monomeric forms (lane 4). Using a 80° C. heat-denaturation step, both the ˜500 kDa and monomeric forms were detectable in similar proportion (lane 3).

In order to investigate further whether this ˜500 kDa species represents an S protein oligomer in native conformation, comparative analyses with virion-derived S glycoprotein derived from Vero cell cultures was performed. The purified virions were solubilised in 1% SDS prior to Western blot analyses after SDS PAGE. The presence of the ˜500 kDa spike protein oligomer was confirmed in virion particles (FIG. 79, lane 1). In addition, heat denaturation of solubilised virions produced the same oligomer-to-monomer conversion as seen with the full-length recombinant S (lanes 2,3). The oligomeric nature of virion S was further analysed in a cross-linking experiment. Aliquots of inactivated virion from sucrose gradient fractions were treated with 10% SDS at 1% final concentration and diluted 2-fold with 0.2M Triethanolamine-HCl (pH 8, Sigma); Dimethyl suberimidate (DMS; Pierce Chemical Co.) was then added from a freshly prepared solution (10 mg/ml in 0.2M Triethanolamine-HCl) at 3.3 mg/ml final concentration. After 2 hr at room temperature, samples were concentrated with Centricon-30 and analyzed by silver staining after electrophoresis on a 4% polyacrylamide gel. Both untreated and DMS cross-linked virion proteins were heat-denatured, and the heat effect on the maintenance of oligomer structure was analysed by SDS-PAGE and silver staining (FIG. 80). In the absence of cross-linking, heat denaturation resulted in the replacement of the ˜500 kD spike protein species with the monomer species. In contrast, in the cross-linked proteins, the levels of the ˜500 kD and monomer species did not change significantly after heating. These data support the fact that the ˜500 kD protein is an oligomer of S monomer proteins that are bound non-covalently. After cross-linking and boiling, the ˜500 kDa species migrated as a somewhat slower diffuse form than the untreated form. This mobility shift is probably due to a structural change resulting from boiling. In addition, a minor protein species of ˜300 kD, which may represent a non-dissociated S dimer, could be seen.

To estimate more precisely the size of the recombinant ˜500 kDa S species expressed in COS7 cells, a COS7 cell lysate containing the S protein oligomer was fractionated using size-exclusion column chromatography. The major portion of the ˜500 kDa oligomer co-eluted with a 572 kDa marker protein. Taken together, these experiments suggest that the ˜500 kDa S species seen in COS7 cell lysates is probably a homotrimer of the S protein monomer.

The oligomeric status of the SΔ spike protein was also examined after expression in COS7 cells. As shown in FIG. 81, the recombinant SΔ proteins present in cell lysates were also detected in high molecular weight forms of ˜500 kDa range when the lysate was not heated prior to SDS-PAGE and Western blot analysis (lane 1). However, the efficiency of oligomerization by intracellular SΔ protein appears to be much less (<10%) compared to that of full-length S protein under the same western analysis conditions. A heat-sensitivity test on this ˜500 kDa protein showed that the SΔ oligomer was more heat labile than that of the full-length S oligomer, as demonstrated by the >90% conversion of all of the ˜500 kDa species into monomeric Sd forms at 80° C. (lane 2). Also (FIG. 82), the majority of the secreted SΔ protein was found in monomeric form with the ˜500 kDa species barely detectable (and only detectable when the protein was loaded in excess for Western analysis) (lane 1). At a temperature above 80° C., all secreted SΔ proteins were detected as monomers (lanes 2, 3).

The ˜500kDa protein is glycosylated, and the effect of deglycosylation on its antibody binding was investigated. The recombinant COS7 lysate was treated with PNGase F under non-denaturing condition (as described above) and analysed by western blot. As shown in FIG. 83, deglycosylation did not affect the binding of anti-histidine Mab antibody to the treated S oligomer (lanes 2,3). However, it compromised the reactivity with the rabbit antisera raised against purified virus (lane 6). This antiserum binds to virion-derived S in western blot analyses only when DTT is omitted from the sample for SDS-PAGE indicating that it recognizes primarily a discontinuous, conformational epitope(s). This antisera has also been shown to have a high-titer of viral neutralizing antibodies. Its lack of binding to deglycosylated, recombinant S suggests that the carbohydrate actively contributes to the higher order, native structure of the S polypeptide oligomer.

The difference between the recombinant S and SΔ protein is the presence or absence of the TM-and Cys-rich domains at the C-terminus. This difference predicts that full-length S would be found associated with the membrane fraction while Sd would be in the soluble fraction upon lysis of transfected cells. Therefore, nSh- or nShΔTC-transfected cells were lysed under hypotonic conditions and the soluble cytosol fraction was separated from the insoluble membrane fraction by centrifugation (FIG. 48). As shown in FIG. 84, the S protein was found in the membrane fraction (DF) both as a ˜500 kDa and 180/170 kDa species (lane 4) but was not detectable in the soluble cytosol fraction (AF) (lane 3). However, the truncated SΔ protein was found as a monomeric species (gp170) in both fractions (lanes 5,6). This indicates that the C-terminal TM and Cys-rich domains are required for the anchorage of the S protein to cell membrane.

The cellular location of the S and SΔ proteins in COS7 cells was analyzed by indirect immunofluorescence microscopy. At 48 hr post-transfection, cells were directly fixed with 2% paraformaldehyde without detergent for cell surface staining or treated with detergent followed by Cytofix/Cytoperm solution for intracellular staining. Fixed cells were then stained with rabbit anti-SARS sera (2BE) and FITC-conjugated antibody. The nSh-transfected cells showed foci of S protein indicative of Golgi-localisation (FIG. 85A), while the nShΔTC-transfected cells displayed a uniform distribution of SΔ protein throughout the cytoplasm indicative of ER localisation (FIG. 85B). While the complete S protein was also observed on the surface of transfected cells in unfixed cells (FIG. 85D), the SΔ was undetectable on the cell surface (FIG. 85E). These results indicate the role played by the TM-and Cys-rich domains in anchoring the S protein to the plasma membrane. Although the TM-region alone is likely responsible for membrane anchorage, the potential role played by the Cys-rich region remains to be determined.

The SARS recombinant full-length S protein is thus an N-linked glycoprotein with an estimated molecular weight of 170-180,000 kDa. Deglycosylation with PNGase F resulted in a polypeptide of the expected size for the uncleaved, encoded polypeptide (140 kDa). Both transient and stable expression of the full-length SARS-CoV S gene in a variety of mammalian cells, including COS7, 293, BHK21, and Huh7 cell lines, consistently produced a S protein doublet (gp170/180) as detected in western blot analyses. Pulse-chase analyses of transfected cells demonstrated that the SARS CoV S protein was initially synthesized as an Endo H sensitive gp170 species followed by the gradual appearance of an Endo H resistant gp180 form, presumably as a result of the addition of complex carbohydrate within the Golgi apparatus.

The recombinant S protein was not secreted into the cell culture medium unless the C-terminal 60 amino acids containing the TM-region and the Cys-rich tail were deleted.

The quaternary structure of the full-length recombinant S protein was investigated using cross-linking treatment, heat-denaturation, and size fractionation analyses. The results data are consistent with the recombinant S protein existing as a homotrimer of ˜500 kDa. Similar analyses of virion-derived S yielded the same results. Such a trimeric structure has been reported for other enveloped RNA viruses: the hemagglutinin HA of influenza virus, the E1-E2 heterodimer of alphaviruses and the G protein of vesicular stomatitis virus. Incubations under reducing conditions indicate that the SARS-CoV S trimeric structure is non-covalently associated, and is very stable. S oligomers present in the cell lysate were shown to be resistant to reduction by 10 mM DTT, detergent treatment with 1% SDS, and heat denaturation at up to 60° C. . Incubation at a temperature higher than >80° C. resulted in the dissociation of the trimeric complex as evidenced by the decrease in trimer with the concomitant increase in the monomer bands. The temperature-induced appearance of the high-mannosylated gp170 (ER monomer form) as well as the complex-glycosylated gp180 (Golgi monomer form) suggests that trimerization can occur before the transport of the monomer spike protein to the medial Golgi apparatus. This is consistent with other reports for TGEV, influenza virus HA, and vesicular stomatitis virus G proteins. With these proteins, trimerization was reported to take place before addition of complex oligosaccharides in the Golgi apparatus.

The C-terminally truncated form of S was found in the cell lysate in both oligomeric and monomeric forms at a frequency of 10% and 90%, respectively. The truncated protein secreted into medium was found fully glysosylated and it was essentially all in monomeric form. We conclude that the C-terminal 60 amino acids of the S glycoprotein contains a membrane anchor region that affects the efficiency of trimerization. In S protein trimerization, it is possible that the C-terminal region is required to initiate the event and the triple-stranded coiled coil structures in the S2 stalk domain provide further stabilizing force as seen in HA oligomer of influenza virus.

Example 24 CHO Cells for Spike Protein Expression

CHO cell lines that stably express either the full-length or truncated SARS-CoV spike proteins were prepared. Several stably transfected CHO cell lines were obtained, and FIG. 73 shows western blot data from a panel of representative clones.

Example 25 Expression in E.coli

All SARS-CoV ORFs (FIG. 17, Table 10) were cloned in the pET vector and expressed as C-terminal His-Tag fusion proteins in E. coli. The proteins smaller than 16 KD were also expressed as N-terminal GST (Glutathione S-transferase) fusion proteins using pGEX vector.

Nsp1 and Nsp2, the two SARS-CoV proteins with proteolytic activity, were not expressed as full length proteins due to toxicity in E. coli. The respective genes were instead cloned in different portions in order to separate the catalytic residues (Cys833/His994 for Nsp1; His41/Cys145 for Nsp2) in the resulting recombinant proteins: Nsp1A from nucleotides 2719-5214 of AY310120; Nsp1B from nucleotides 5218-7371; Nsp1C from nucleotide 7372-9984; Nsp2A from nucleotide 9985-10416; Nsp2B from nucleotide 10476-10902.

Nsp9 (SEQ ID NO: 9775) was divided into two portions: Nsp9A from nucleotide 13371-14756; Nsp9B from nucleotide 14757-16166.

Matrix (M), ORF3 and ORF7 contain respectively three, two and one transmembrane domains. These proteins were expressed as deletion proteins excluding the first 100 amino acids (M and ORF3) or the first 18 amino acids (ORF7) that include the hydrophobic regions.

The cloned sequences are shown in Table 26.

A two-step strategy was used to amplify the cloned sequences. In the first step, amplification of DNA fragments containing more than one gene or single gene used sequenced cDNA as template. Eleven cDNA sequences were amplified: (1) a fragment, named amplC1, including genes coding for protein E, protein M, orf 7-8-9-10; (2) a fragment, named amplC2, including genes coding for orf 3-4; (3) a fragment, named amplC5, including genes coding for proteins Nsp12 and Nsp13; (4) Nsp11gene; (5) P28 and P65 genes; (6) Nsp1B and Nsp1C genes portion; (7) a fragment, named amplC9, including genes coding for proteins Nsp2 and Nsp3; (8) a fragment, named amplNsp4-7, including genes coding for proteins Nsp4, Nsp5, Nsp6, Nsp7 and for amplification of Nsp9A gene portion; (9) Nsp 9B gene portion and Nsp10 gene; (10) a fragment, named amplCO, including genes coding for proteins Orf11, Nucleocapsid (N) and Orf12; (11) Nsp1A gene portion. The primers used in this first step are given in Table 27:

In the second step, amplification of single genes was performed using DNA fragments from the first amplification step as templates. The primers are shown in Table 28.

Of the proteins where expression was seen, it was either in inclusion bodies (insoluble) or in a soluble form. Purification proceeded on appropriate material. Table 29 shows the molecular weight of the expressed fragments of SARS-CoV ORFs, whether they were cloned (+ or −), whether the cloned fragment was seen to be expressed (+ or −) and the form of protein which was chosen for purification.

Where a protein was a soluble His-tagged product, a single colony was streaked and grown overnight at 37° C. on a LB/Amp (100 μg/ml) agar plate. An isolated colony from this plate was inoculated into 20 ml of LB/Amp (100 μg/ml) liquid medium and grown overnight at 37° C. with shaking. The overnight culture was diluted 1:30 into 1.0 L LB/Amp (100 μg/ml) liquid medium and allowed to grow at the optimal temperature (30 or 37° C.) until the OD550 nm reached 0.6-0.8. Expression of recombinant protein was induced by addition of IPTG (final concentration 1.0 mM) and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 8000×g for 15 min at 4° C. The bacterial pellet was resuspended in 10 ml of cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8.0). Cells were disrupted by sonication (or French Press) on ice four times for 30 sec at 40 W using a Branson sonifier 450 and centrifuged at 13 000× g for 30 min at 4° C. Supernatants were mixed with 150 μl Ni²⁺-resin (previously equilibrated with buffer A) and incubated at room temperature with gentle agitation for 30 min. The resin was Chelating Sepharose Fast Flow (Pharmacia), prepared according to the manufacturer's protocol. The batch-wise preparation was centrifuged at 700×g for 5 min at 4° C. and the supernatant discarded. The resin was washed twice (batch-wise) with 10 ml buffer A for 10 min, resuspended in 1.0 ml buffer A and loaded onto a disposable column. The resin continued to be washed with buffer A at 4° C. until the OD280nm of the flow-through reached 0.02-0.01. The resin was further washed with cold buffer B(300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8.0) until the the OD280 nm of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700 μl of cold elution buffer C (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8.0) and fractions collected until the OD_(280mm) indicated all the recombinant protein was obtained. 20 μl aliquots of each elution fraction were analyzed by SDS-PAGE. Protein concentrations were estimated using the Bradford assay.

Where a protein was seen as an insoluble product, the inclusion bodies were purified as follows: homogenize cells (5 g wet weight) in 25 ml 0.1M Tris HCl pH 7, 1 mM EDTA, at 4° C. using an ultraturrax (10000 rpm); add 1.5 mg lysozyme per gram cells; mix shortly with an ultraturrax and incubate at 4° C. for 30′; use sonication or high-pressure homogenization to disrupt the cells; to digest DNA, add MgCl₂ to a final concentration of 3 mM and DNase to a final concentration of 10 ug/ml and incubate 30′ at 25° C. add 0.5 vol of 60 mM EDTA, 6% Triton x-100, 1.5M NaCl pH 7.0 to the solution, and incubate for 30′ at 4° C.; centrifugation at 31000 g for 10′ at 4° C.; re-suspend pellet in 40 ml of 0.1M Tris HCl pH 7.0, 20 mM EDTA using ultraturrax; centrifugation at 31000 g for 10′ a 4° C.; store the IB pellet at −20° C.

The results of expression are shown in FIGS. 86 to 105. Examples of purity and yield are given in Table 30.

Example 26 Retention of Critical Epitope on Truncated Spike Antigen

A human monoclonal antibody having neutralizing activity was tested in an ELISA assay for reactivity with the purified truncated Spike protein. Briefly, ELISA plates were coated with truncated form of the spike protein at a concentration of 1 μg/ml (100 μ/well) by incubating the plates overnight at 4° C. The plates were washed, non-specific binding sites were blocked and then different dilutions of the antibody were added and plates were incubated for 1 hour at room temperature. At the end of incubation, the plates were washed and bound antibody was detected by using anti-human IgG conjugated to horse radish peroxidase (HRP) and an appropriate substrate. The optical density of each well was recorded at 405 nm using an ELISA reader. The data are shown in FIG. 69 and clearly demonstrate that the neutralizing epitope recognized by the mAb is preserved and exposed on the recombinant truncated Spike protein.

Example 27 Different Spike Vaccines

Purified truncated spike protein was used to immunize mice and the level of binding antibodies induced against the truncated spike protein was determined by ELISA assay. Briefly a group of 10 mice were immunized with 3 μg of truncated spike protein adjuvanted in MF59 at 0, 4 and 8 weeks intervals. Sera samples were collected from these animals and assayed for antibodies induced by truncated spike protein in an ELISA assay. An additional group of 8 mice was immunized with 75 jig of DNA encoding the truncated form of the spike protein on PLG particles at 0, 4 and 13 weeks intervals, the sera were collected and analyzed as above for anti-spike antibodies as above

The profile of binding antibodies induced in each group was plotted as geometric mean titer (GMT). Compared to a plasmid DNA vaccine expressing truncated spike antigen and delivered using a PLG microparticle formulation, the purified truncated spike protein was significantly more potent for inducing strong antibody responses. Further comparison with the antibody responses induced by inactivated BPL-SARS-CoV (already shown protective) in the same mouse strain revealed that the magnitude of antibody responses induced by purified truncated spike protein and the inactivated virus vaccine are in the same range (FIG. 70).

The neutralization potential of antibodies induced by the recombinant truncated spike protein, or plasmid DNA expressing the same spike antigen, were also evaluated. The GMT values obtained in both groups are shown in FIG. 71. From these data, it appears that the purified protein is significantly more effective at inducing neutralizing antibody responses against the SARS-CoV spike. Furthermore, the neutralization titers typically induced by the purified truncated spike protein are comparable to neutralization titers induced by an inactivated SARS-CoV vaccine.

FIG. 72 shows a comparison of antibody binding levels (ELISA, X-axis) with neutralization titers (Y-axis). In general there is a very good correlation between the binding and neutralizing antibodies. The bottom-left grouping shows ratios 2 weeks post-3rd immunization with the DNA vaccine; the top-right grouping shows ratios 2 weeks post-2nd immunization with the protein vaccine. Both forms of vaccine show a consistent correlation.

In further experiments, the ability of a DNA vaccine to invoke an immune response in mice was studied. Mice were immunized with pCMV-nSdTC plasmid, either free or with PLG microparticles. Serum from the mice was then used as the staining antibody against cultured 293 cells that had been transfected with spike, either full-length or truncated. The cells were centrifuged prior to testing and the pellet was lysed. The antibody was tested against the culture supernatant and against the cell lysate. As shown in FIG. 112, the mouse serum was able to detect spike protein in the lysate of cells that expressed full-length spike and in the supernatant of cells that expressed the truncated spike protein. Results were comparable to the staining seen when using rabbit serum that had been obtained after immunization with whole killed virus. Thus anti-spike antibodies can be induced by the use of DNA vaccination.

Example 28 Expression Cassettes in pCMV

The sequence of plasmid pCMVKm2 is given as SEQ ID NO: 9923. Genes encoding the spike protein either in full-length form (pCMVKm2 SARS Spike nS; SEQ ID NO: 9921) or in its ΔTC form (pCMVKm2 SARS Spike nSΔTC; SEQ ID NO: 9922) were inserted into this basic vector.

Mice were immunized with these vectors, and with similar vectors encoding the N, M or E proteins. Vectors encoding the same proteins but with optimized codon usage were also prepared. Codons were optimized for efficient human expression starting from the FRA sequence (GenBank: AY310120). The optimized sequences are: N (SEQ ID NO: 9924); M (SEQ ID NO: 9925); E (SEQ ID NO: 9926).

After administration, expression of proteins could be detected by immunofluorescence in all cases. For example, FIG. 106 shows immunofluorescence (using anti-SARS rabbit serum) results after administration of the vector encoding optimsed N antigen, revealing high level expression. Mice receiving the control vector alone showed no fluorescence.

FIG. 107 compares immunofluorescence (using Abgent anti-M antibody) of the native M sequence (107A) or the codon-optimsed M sequence (107B). Similarly, FIG. 108 compares immunofluorescence (using Abgent anti-E antibody) of the native E sequence (108A) or the codon-optimsed E sequence (108B).

Four groups of mice (8 mice per group) were immunized with: (1) SARS nS Spike, nSdTC truncated Spike, and N proteins; (2) pCMV-SARS-nSdTC: DNA+DNA-PLG at weeks 0,4 and 13 wks; (3) CMV-nS: DNA+DNA-PLG+VEE/SIN Rep at 0, 4 and 9 wks; (4) VEE/SIN Rep-SARS-nS three times at 0, 4 and 13 wks. Sera from all groups recognized SARS nS and nSdTC proteins, and also showed virus binding and neutralization activity.

Example 29 Spike Protein Cleavage

To investigate the effect of proteolytic cleavage on SARS-CoV Spike protein, it was expressed in various forms in E.coli, including: (1) full-length S1-S2; (2) S1 alone; (3) HR1 heptad; and (4) HR2 heptad. The expressed proteins were used to raise immune rabbit sera, which were then used for visualizing western blots of Vero cells, either infected or not infected with SARS-CoV.

FIG. 109 shows a western blot using a 1:10000 dilution of antibody raised against either the S1 domain or the uncleaved S1-S2 domains. FIG. 110 shows a western blot using a 1:10000 dilution of antibody raised against each of the four proteins. The difference in antigen reactivity is readily apparent.

FIG. 111 shows similar data. Each serum was tested against four lanes, with those gour lanes being from left to right: (a) serum at 1:500 dilution, SARS-CoV-infected cells; (b) serum at 1:500 dilution, non-infected cells; (c) serum at 1:2500 dilution, SARS-CoV-infected cells; (d) serum at 1:2500 dilution, non-infected cells. Again, the difference in antigen reactivity is readily apparent.

FIGS. 109-111 show that the Spike protein exists in various forms in infected Vero cells, with sizes of approx. 75 kDa, 90 kDa, 180 kDa and >250 kDa. The Spike protein is cleaved (at least partially) either intracellulary or after release of the particles.

If enzymatic cleavage of the mouse hepatitis coronavirus spike protein is inhibited then cell-cell fusion (syncytia formation) is also inhibited, but virus-cell fusion is not (de Haan et al. (2004) J Virol). Syncytia are observed in vivo in the lungs of SARS-infected patients, but are not seen in Vero cell cultures of the SARS-CoV. Inhibition of Spike protein cleavage may thus be used to prevent syncytia formation and related pathology, even though viral infectivity may not be blocked.

Example 30 Purification of SARS Protease

Cells were grown at 37° C. to mid-log phase and induced with 0.2% L-arabinose. Cells were harvested by centrifugation, and the cells resuspended in lysis buffer (LB) containing 20 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol V/V, 0.05% Triton X-100, 5 mM βME, 5 mM imidazole, and complete protease inhibitors (−)EDTA. Benzonase was added to a final concentration of 50 U/ml of lysate. Cells were then lysed using two passes through a pre-chilled microfluidizer. The lysate was clarified by high speed centrifugation at 44,000× g. Clarified lysate was applied to a prepared Pharmacia chelating FF column charged with nickel sulfate. After application of the lysate the column was washed with 5 column volumes of LB, followed by 5 column volumes of LB supplemented with 45 mM imidazole. The column was then eluted using LB supplemented with 250 mM imidazole. Purity of the isolated SARS protease was 50%. Fractions containing protease were pooled, adjusted to 5 mM EDTA, and then applied to a Superdex 200 gel filtration column equilibrated in 20 mM Tris pH 7.5, 150 mM NaCl, 5% V/V glycerol, 0.05% Triton X-100, and 5 mM DTT. Purity of the isolated SARS protease was 70%. Again, fractions containing the protease were pooled, and then stored at −80° C. until used. Activity assay, mass spectrometry and western blot analysis were used to positively identify the protein (FIG. 133). All steps were carried out with pre-chilled buffers, and kept at 4° C. for as much of the preparation as possible.

Western of SARS Protease Purification Fractions

Protocol: Briefly, protein concentration was based on Absorbance at 280 nm, and coomassie stained gel estimates of purity. Protein was run on a 4-20% gradient gel, and transferred to nitrocellulose. The blot was then blocked with 3% BSA, probed with Mouse IgG anti-pentaHis, and then probed with a secondary antibody to Mouse IgG conjugated with HRP. The blot was visualized using an ECL kit (Pharmacia Biotech). The results are shown in FIG. 133 where A is the sizing column pool loaded at 50, 100 and 200 ng of target protein and B is the immobilized metal affinity column pool loaded at 50, 100 and 200 ng of target protein.

Example 31 Continuous Fluorescence Resonance Energy Transfer (FRET) Enzyme Assay

The peptide containing EDANS, the fluorescence donor, and DABCYL, the fluorescence quencher (DABCYL-VNSTLQ ∇SGLRK-EDANS) was synthesized by Syn. Pep. (Dublin, Calif.). The peptide contains the cleavage site Gln-Ser in the middle. Meyers, G. et al. Handbook of Proteolytic Enzymes and Barrett, A et al., Academic Press, London, 1998, 726-728. The proteolytic activity of SARS protease was followed kinetically by measuring the level of formation of cleaved product that contains the fluorescence donor, SGLRK-EDANS using the Hitachi fluorometer (F-4500 FL Spec.) set at 340 nm excitation and 490 nm emission wave length. 5 μL of 5 mM peptide stock in DMSO solution was added to the reaction mixture, containing 295 μl of standard buffer (75 mM Tris-Hcl, 25 mM NaOAc, 25 mM Bis-Tris, 25 mM glycine, 5 mM EDTA, and 1 mM EDTA, pH 7.4) and 100 ul of buffer or 100 ul of 3.6 uM protease stock solution. The kinetic curve was followed for 6 minutes (the reaction was linear with R2 value of 0.998 (FIG. 134)). The formation of fluorescence (proteolytic reaction) is likely enzyme dependent, as concentration of enzyme was tripled three times as much fluorescence was formed in the 6 minutes time frame.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. TABLE 1 US Patents and Published International Patent Applications Publication Publication Number Title Date US-3927216 1,2,4-Triazol E-3-Carboxamides For Inhibiting Virus Infections Dec. 16, 1975 US-4010269 Antiviral Quinazoline Compositions And Methods Of Use Mar. 1, 1977 US-4065570 Antiviral 5-(Substituted Benzal) Hydantoins Dec. 27, 1977 US-4089965 Thiazolylphenylguanidines As Antirhinovirus Agents May 16, 1978 US-4122191 Antirhinovirus Agents Oct. 24, 1978 US-4192895 Antirhinovirus Agents Mar. 11, 1980 US-4254144 Substituted Benzonitriles Having Antiviral Activity Mar. 3, 1981 US-4264617 Antiviral 5-(Substituted Benzal) Hydantoins Apr. 28, 1981 US-4287188 Purine Derivatives Sep. 1, 1981 US-4327088 Phosphonooxy- Or Glycosyloxy-Substituted Acrylophenones, Compositions And Uses Thereof Apr. 27, 1982 US-4332820 Substituted Benzonitriles Having Antiviral Activity Jun. 1, 1982 US-4349568 Sulfur-Substituted Diphenyl Ethers Having Antiviral Activity Sep. 14, 1982 US-4352792 3-Alkoxyflavone Antiviral Agents Oct. 5, 1982 US-4371537 Sulfur-Substituted Phenoxypyridines Having Antiviral Activity Feb. 1, 1983 US-4423053 Derivatives Of 2-Amino-5-(O-Sulphamidophenyl)-1,3,4-Thiadiazol As Antiviral Agents And A Dec. 27, 1983 Process For The Preparation Thereof US-4505929 Sulfur-Substituted Diphenyl Ethers Having Antiviral Activity Mar. 19, 1985 US-4526897 Hypertensive Isoindolin-2-Yl-Aminoimidazolines And Isoindolin-2-Yl-Guanidines Jul. 2, 1985 US-4558134 Certain Phenoxy-Pyridine-Carbonitriles Having Antiviral Activity Dec. 10, 1985 US-4629729 Endowed With Anti-Viral Activity 2-Alkylamino-4,6-Dihalo Pyrimidines Dec. 16, 1986 US-4636492 Inhibition Of Viral Protease Activity By Peptide Halomethyl Ketones Jan. 13, 1987 US-4652552 Tetrapeptide Methyl Ketone Inhibitors Of Viral Proteases Mar. 24, 1987 US-4724233 Therapeutical Application Of Phosphonylmethoxyalkyl Adenines Feb. 9, 1988 US-4738984 Antirhinovirus Agents Apr. 19, 1988 US-4847246 Antiviral Compositions Derived From Fireflies And Their Methods Of Use Jul. 11, 1989 US-4855283 Novel Pharmaceutically Active N-(2-Aminoacylamido-2-Deoxy-Hexosyl)-Amides, -Carbamates Aug. 8, 1989 And -Ureas US-4885285 Phosphorus Compounds, Processes For Their Manufacture, And Their Use Dec. 5, 1989 US-4956351 Antiviral Pharmaceutical Compositions Containing Cyclodextrins Sep. 11, 1990 US-5001125 Anti-Virally Active Pyridazinamines Mar. 19, 1991 US-5036072 Antiviral Agent Jul. 30, 1991 US-5070090 Antipicorpaviral Herterocyclic-Substituted Morpholinyl Alkylphenol Ethers Dec. 3, 1991 US-5100893 Antipicornaviral Pyridazinamines Mar. 31, 1992 US-5112825 Antirhinoviral Heteroamine-Substituted Pyridazines May 12, 1992 US-5157035 Anti-Virally Active Pyridazinaniines Oct. 20, 1992 US-5240694 Combined Antiviral And Antimediator Treatment Of Common Colds Aug. 31, 1993 US-5242924 Tetrazolyl-(Phenoxy And Phenoxyalkyl)-Piperidinylpyridazines As Antiviral Agents Sep. 7, 1993 US-5278184 Synthetic Derivatives Of Pyrrole And Pyrrolidine Suitable For The Therapy Of Infections Caused Jan. 11, 1994 By Rhinoviruses US-5364865 Phenoxy- And Phenoxyalkyl-Piperidines As Antiviral Agents Nov. 15, 1994 US-5453433 Thiadiazoles And Antipicornaviral Compositions Sep. 26, 1995 US-5492689 Combined Virustatic Antimediator (COVAM) Treatment Of Common Colds Feb. 20, 1996 US-5514679 Therapeutic Phenoxyalklpyridazines And Intermediates Therefor May 7, 1996 US-5514692 Substituted Quinoline Derivatives Useful As Antipiconaviral Agents May 7, 1996 US-5523312 Antipicornaviral Agents Jun. 4, 1996 US-5545653 Anti-Viral Compounds Aug. 13, 1996 US-5552420 Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines Sep. 3, 1996 US-5567719 Thiadiazoles And Their Use As Antipicornaviral Agents Oct. 22, 1996 US-5580897 1,2-Dithiins Having Antifungal Activity Dec. 3, 1996 US-5618821 Therapeutic Phenoxyalkylheterocycles Apr. 8, 1997 US-5618849 Orally Active Antiviral Compounds Apr. 8, 1997 US-5648354 1,2-Dithiins Having Antifungal Activity Jul. 15, 1997 US-5650419 Thiadiazoles And Their Use As Antipicornaviral Agents Jul. 22, 1997 US-5693661 Anti-Viral Compounds Dec. 2, 1997 US-5721261 Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines Feb. 24, 1998 US-5725859 Plant-Based Therapeutic Agent With Virustatic And Antiviral Effect Mar. 10, 1998 US-5750527 Thiadiazoles And Their Use As Antipicornaviral Agents May 12, 1998 US-5750551 Treatment For Viral Diseases May 12, 1998 US-5762940 Methods And Compositions For Inhibiting Or Destroying Viruses Or Retroviruses Jun. 9, 1998 US-5763461 Therapeutic Phenoxyalkylheterocycles Jun. 9, 1998 US-5821242 Anti-Viral Compounds Oct. 13, 1998 US-5821257 Thiadiazoles And Their Uses As Antipicornaviral Agents Oct. 13, 1998 US-5821331 Anti-Picornaviral Agents Oct. 13, 1998 US-5846986 Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines Dec. 8, 1998 US-5856530 Antipicornaviral Compounds And Methods For Their Use And Preparation Jan. 5, 1999 US-5891874 Anti-Viral Compound Apr. 6, 1999 US-5962487 Antipicornaviral Compounds And Methods For Their Use And Preparation Oct. 5, 1999 US-6004933 Cysteine Protease Inhibitors Dec. 21, 1999 US-6020371 Antipicornaviral Compounds Compositions Containing Them And Methods For Their Use Feb. 1, 2000 US-6087374 Anti-Viral Compounds Jul. 11, 2000 US-6114327 Anti-Viral Compounds Sep. 5, 2000 US-6117844 Method And Composition For Antiviral Therapy Sep. 12, 2000 US-6194447 Bis (Benzimidazole) Derivatives Serving As Potassium Blocking Agents Feb. 27, 2001 US-6214799 Antipicornaviral Compounds And Methods For Their Use And Preparation Apr. 10, 2001 US-6277891 Nitric Oxide Inhibits Rhinovirus Infection Aug. 21, 2001 US-6294186 Antimicrobial Compositions Comprising A Benzoic Acid Analog And A Metal Salt Sep. 25, 2001 US-6331554 Antipicornaviral Compounds, Compositions Containing Them, And Methods For Their Use Dec. 18, 2001 US-6358971 Anti-Viral Compounds Mar. 19, 2002 US-6362166 Antipicornaviral Compounds And Methods For Their Use And Preparation Mar. 26, 2002 US-6414004 3-Substituted 5-Aryl-4-Isoxazolecarbonitriles Having Antiviral Activity Jul. 2, 2002 US-6420591 Carbamates And Compositions Thereof, And Methods For Their Use For Treating Cancer, Jul. 16, 2002 Inflammation, Or A Viral Infection US-6469018 Compounds Oct. 22, 2002 US-6498178 Inhibitors Of IMPDH Enzyme Dec. 24, 2002 US-6514997 Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Feb. 4, 2003 Their Synthesis US-6525043 Use Of Ion Channel Modulating Agents Feb. 25, 2003 US-6531452 Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Mar. 11, 2003 Their Synthesis US-6534489 Organophosphorus Compounds And The Use Thereof Mar. 18, 2003 WO 00/06529 Diketoacid-Derivatives As Inhibitors Of Polymerases Feb. 10, 2000 WO 00/25791 Pyridin-4-Yl Or Pyrimidin-4-Yl Substituted Pyrazines May 11, 2000 WO 00/27423 Methods And Compositions For Treating Common Cold Symptoms May 18, 2000 WO 00/34308 Protein Transduction System And Methods Of Use Thereof Jun. 15, 2000 WO 00/39348 Methods And Compositions For Identifying Protease Modulators Jul. 6, 2000 WO 00/40243 Novel Compounds Jul. 13, 2000 WO 00/50037 Nitrosated And Nitrosylated Proton Pump Inhibitors, Compositions And Methods Of Use Aug. 31, 2000 WO 00/56331 Inhibitors Of Impdh Enzyme Sep. 28, 2000 WO 00/56757 Immunomodulatory Steroids, In Particular The Hemihydrate Of 16.Alpha.-Bromoepiandrosterone Sep. 28, 2000 WO 00/66096 New Indication For Use Of Antiepileptic Agents And Medicines Nov. 9, 2000 WO 00/78746 Antiviral Agents Dec. 28, 2000 WO 01/00199 Compounds Obtained From Salvia Species Having Antiviral Activity Jan. 4, 2001 WO 01/00585 Pyrazolidinol Compounds Jan. 4, 2001 WO 01/02551 Virus Like Particles, Their Preparation And Their Use Preferably In Pharmaceutical Screening Jan. 11, 2001 And Functional Genomics WO 01/03681 Use Of Flavones, Coumarins And Related Compounds To Treat Infections Jan. 18, 2001 WO 01/05396 Use Of Cobalt Chelates For Treating Or Preventing Virus Infection Jan. 25, 2001 WO 01/10894 Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Feb. 15, 2001 Their Synthesis WO 01/19322 Use Of Csaids In Rhinovirus Infection Mar. 22, 2001 WO 01/19822 Antiviral Agents Mar. 22, 2001 WO 01/22920 Colon And Colon Cancer Associated Polynucleotides And Polypeptides Apr. 5, 2001 WO 01/25188 Novel Carbamates And Ureas Apr. 12, 2001 WO 01/31016 Processed Human Chemokines Phc-1 And Phc-2 May 3, 2001 WO 01/37837 3,4-Dihydro-(1h)-Quinazolin-2-Ones And Their Use As Csbp/P38 Kinase Inhibitors May 31, 2001 WO 01/38312 3,4-Dihydro-(1h)Quinazolin-2-One Compounds As Csbp/P38 Kinase Inhibitors May 31, 2001 WO 01/38313 3,4-Dihydro-(1h)Quinazolin-2-One Compounds As Csbp/P39 Kinase Inhibitors May 31, 2001 WO 01/38314 3,4-Dihydro-(1h)Quinazolin-2-One Compounds As Csbp/P38 Kinase Inhibitors May 31, 2001 WO 01/40189 Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Jun. 7, 2001 Their Synthesis WO 01/49303 Multivalent Electron Active Compositions And Methods Of Making And Using Same Jul. 12, 2001 WO 01/60393 Selective Destruction Of Cells Infected With Human Immunodeficiency Virus Aug. 23, 2001 WO 01/62726 2-Oxo-1-Pyrrolidine Derivatives, Processes For Preparing Them And Their Uses Aug. 30, 2001 WO 01/79167 Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Oct. 25, 2001 Their Synthesis WO 01/90047 Novel Mmp-2/Mmp-9 Inhibitors Nov. 29, 2001 WO 01/90129 Prophylactic And Therapeutic Treatment Of Infectious And Other Diseases With Mono- And Nov. 29, 2001 Disaccharide-Based Compounds WO 01/92499 Nucleic Acid Molecules Encoding A Protein Interacting With Ser/Thr Kinase Akt Dec. 6, 2001 WO 01/93883 Therapeutic Agents - Iii Dec. 13, 2001 WO 01/93884 Therapeutic Agents - I Dec. 13, 2001 WO 01/93885 Therapeutic Agents - Ii Dec. 13, 2001 WO 01/96297 Antipicornaviral Compounds And Compositions, Their Pharmaceutical Uses, And Materials For Dec. 20, 2001 Their Synthesis WO 02/04413 Chiral Integrin Modulators And Methods Of Use Thereof Jan. 17, 2002 WO 02/11743 Treatment Of Prostate Cancer Feb. 14, 2002 WO 02/12477 Enhanced Replication Of Hcv Rna Feb. 14, 2002 WO 02/14343 Immunosuppressive, Antiinflammatory And Analgetic Compounds Feb. 21, 2002 WO 02/24145 Antiviral Substances From Plant Cuticular And Epicuticular Material Mar. 28, 2002 WO 02/28351 Recombinant Mucin Binding Proteins From Steptococcus Pneumoniae Apr. 11, 2002 WO 02/30442 Method For Treating Cytokine Mediated Hepatic Injury Apr. 18, 2002 WO 02/34771 Nucleic Acids And Proteins From Streptococcus Groups A & B May 2, 2002 WO 02/44737 Diagnostic And Therapeutic Compositions And Methods Related To G Protein-Coupled Receptor Jun. 6, 2002 (Gpcr) Anaphylatoxin C3a Receptor WO 02/50045 Antiviral Agents Jun. 27, 2002 WO 02/51413 Macrocyclic Anti-Viral Compounds Jul. 4, 2002 WO 02/53138 Treatment For Inhibiting Neoplastic Lesions Jul. 11, 2002 WO 02/57425 Nucleoside Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase Jul. 25, 2002 WO 02/59083 Novel Compounds Aug. 1, 2002 WO 02/60875 Nicotinamide Biaryl Derivatives Useful As Inhibitors Of Pde4 Isozymes Aug. 8, 2002 WO 02/60898 Thiazolyl-, Oxazolyl-, Pyrrolyl-, And Imidazolyl-Acid Amide Derivatives Useful As Inhibitors Aug. 8, 2002 Of Pde4 Isozymes WO 02/69903 Nucleosides, Preparation Thereof And Use As Inhibitors Of Rna Viral Polymerases Sep. 12, 2002 WO 02/72022 Substituted Tetracycline Compounds As Antifungal Agents Sep. 19, 2002 WO 02/72031 Substituted Tetracycline Compounds As Synergistic Antifungal Agents Sep. 19, 2002 WO 02/76939 Cysteine Protease Inhibitors Oct. 3, 2002 WO 02/77021 Streptococcus Pneumoniae Proteins And Nucleic Acids Oct. 3, 2002 WO 02/79401 Novel Rgs9 Protein Binding Interactions And Methods Of Use Thereof Oct. 10, 2002 WO 02/82041 Production And Use Of Novel Peptide-Based Agents For Use With Bi-Specific Antibodies Oct. 17, 2002 WO 02/87465 Compositions And Methods Of Double-Targeting Virus Infections And Cancer Cells Nov. 7, 2002 WO 02/87500 Viral Enzyme Activated Prototoxophores And Use Of Same To Treat Viral Infections Nov. 7, 2002 WO 02/88091 Inhibitors Of Human Rhinovirus 2a Cysteine Protease Nov. 7, 2002 WO 02/89832 Pharmaceutical Compositions For Preventing Or Treating Th1 And Th2 Cell Related Diseases By Nov. 14, 2002 Modulating The Th1/Th2 Ratio. WO 02/92779 Method For Enriching Tissues In Long Chain Polyunsaturated Fatty Acids Nov. 21, 2002 WO 02/94185 Conjugates And Compositions For Cellular Delivery Nov. 28, 2002 WO 02/94868 Staphylococcus Aureus Proteins And Nucleic Acids Nov. 28, 2002 WO 02/96867 Inhibitors Of Protein Kinase For The Treatment Of Disease Dec. 5, 2002 WO 02/98424 Novel Anti-Infectives Dec. 12, 2002 WO 03/04489 Compositions And Methods For Inhibiting Prenyltransferases Jan. 16, 2003 WO 03/08628 Enzymatic Nucleic Acid Peptide Conjugates Jan. 30, 2003 WO 03/15744 Chitin Microparticles And Their Medical Uses Feb. 27, 2003 WO 03/20222 Dioxolane And Oxathiolane Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase Mar. 13, 2003 WO 03/20270 Oxadiazolyl-Phenoxyalkylisoxazoles, Compositions Thereof And Methods For Their Use As Mar. 13, 2003 Anti-Picornaviral Agents WO 03/20271 Oxadiazolyl-Phenoxyalkylisoxazoles, Compositions Thereof And Methods For Their Use As Mar. 13, 2003 Anti-Picornaviral Agents WO 03/20712 Oxadiazolyl-Phenoxyalkylthiadiazoles, Compositions Thereof And Methods For Their Use As Mar. 13, 2003 Anti-Picornaviral Agents WO 86/03412 Improvements Relating To The Treatment Control And Prevention Of Rhinovirus Infections Jun. 19, 1986 WO 86/03971 Antiviral Agents Jul. 17, 1986 WO 88/09669 Avirulent Microbes And Uses Therefor Dec. 15, 1988 WO 92/03475 Enterovirus Peptides Mar. 5, 1992 WO 92/22520 Orally Active Antiviral Compounds Dec. 23, 1992 WO 92/22570 Inhibitors Of Picornavirus Proteases Dec. 23, 1992 WO 94/00012 Nucleic Acids And Methods Of Use Thereof For Controlling Viral Pathogens Jan. 6, 1994 WO 95/03821 Prosaposin And Cytokine-Derived Peptides As Therapeutic Agents Feb. 9, 1995 WO 95/09175 Ring-Expanded Nucleosides And Nucleotides Apr. 6, 1995 WO 95/11992 Antiviral Compounds May 4, 1995 WO 95/31198 Thiadiazoles And Their Use As Antipicornaviral Agents Nov. 23, 1995 WO 95/31438 Therapeutic Phenoxyalkylheterocycles Nov. 23, 1995 WO 95/31439 Therapeutic Phenoxyalkylpyridazines And Intermediates Therefor Nov. 23, 1995 WO 95/31452 Therapeutic Phenoxyalkylazoles And Phenoxyalkylazines Nov. 23, 1995 WO 95/34595 Antiviral Dendrimers Dec. 21, 1995 WO 95/35103 A Pharmaceutical Composition For The Prevention And/Or Treatment Of Viral Infections And Dec. 28, 1995 Optionally Inflammations As Well As A Method For The Treatment Thereof WO 96/05836 Methods Of Treating Cold Symptoms Using Pentoxifylline Feb. 29, 1996 WO 96/05854 Combination Preparation, Containing Cyclosporin A Or Fk506 Or Rapamycin And A Xanthine Feb. 29, 1996 Derivative WO 96/09822 Antipicornaviral Agents Apr. 4, 1996 WO 96/11211 Selective Inhibition Of Internally Initiated Rna Translation Apr. 18, 1996 WO 96/22689 Multiple Component Rna Catalysts And Uses Thereof Aug. 1, 1996 WO 96/40641 Sulfonamide Derivatives As Cell Adhesion Modulators Dec. 19, 1996 WO 97/08553 Targeting Of Proteins To The Cell Wall Of Gram-Positive Bacteria Mar. 6, 1997 WO 97/34566 Electrophilic Ketones For The Treatment Of Herpesvirus Infections Sep. 25, 1997 WO 97/41137 Use Of Anthocyanidin And Anthocyanidin Derivatives Nov. 6, 1997 WO 97/43305 Inhibitors Of Picornavirus 3c Proteases And Methods For Their Use And Preparation Nov. 20, 1997 WO 97/47270 Novel Anti-Viral Compounds Dec. 18, 1997 WO 98/03572 Antiviral Linear Polymers Jan. 29, 1998 WO 98/07745 Compositions And Methods For Treating Infections Using Analogues Of Indolicidin Feb. 26, 1998 WO 98/11778 Antimicrobial Treatment For Herpes Simplex Virus And Other Infectious Diseases Mar. 26, 1998 WO 98/22495 Antikinin Compounds And Uses Thereof May 28, 1998 WO 98/31363 Anti-Viral Compounds Jul. 23, 1998 WO 98/31374 Method Of Treating Rhinoviral Infections Jul. 23, 1998 WO 98/32427 Therapeutic Treatment And Prevention Of Infections With A Bioactive Material Encapsulated Jul. 30, 1998 Within A Biodegradable-Biocompatible Polymeric Matrix WO 98/34601 Method For Inhibiting Intracellular Viral Replication Aug. 13, 1998 WO 98/42188 Antimicrobial Prevention And Treatment Of Human Immunedeficiency Virus And Other Oct. 1, 1998 Infectious Diseases WO 98/43950 Antipicornaviral Compouds, Compositions Containing Them, And Methods For Their Use Oct. 8, 1998 WO 98/49190 Substituted Oxadiazole Cysteine Protease Inhibitors Nov. 5, 1998 WO 98/55120 Anti-Viral Compounds Dec. 10, 1998 WO 99/30699 Modulators Of Cysteine Protease Jun. 24, 1999 WO 99/31122 Antipicornaviral Compounds And Methods For Their Use And Preparation Jun. 24, 1999 WO 99/54317 Cysteine Protease Inhibitors Oct. 28, 1999 WO 99/55663 Inhibitors Of Impdh Enzyme Nov. 4, 1999 WO 99/57135 Antipicornaviral Compounds, Their Preparation And Use Nov. 11, 1999 WO 99/59587 Anti-Viral Compounds Nov. 25, 1999 WO 99/61437 Novel 2-Alkyl Substituted Imidazole Compounds Dec. 2, 1999

TABLE 2 US Patents and Published International Patent Applications Publication Publication Number Title Date WO 02/69903 Nucleosides, Preparation Thereof And Use As Inhibitors Of Rna Viral Polymerases Sep. 12, 2002 WO 02/48116 Inhibitors Of Hepatitis C Virus Ns3 Protease Jun. 20, 2002 WO 02/48157 Imidazolidinones And Their Related Derivatives As Hepatitis C Virus Ns3 Protease Inhibitors Jun. 20, 2002 WO 02/61048 In Vitro System For Replication Of Rna-Dependent Rna Polymerase (Rdrp) Viruses Aug. 8, 2002 WO 03/02518 Novel 2,4-Difluorobenzamide Derivatives As Antiviral Agents Jan. 9, 2003 WO 02/79187 Methoxy-1,3,5-Triazine Derivatives As Antiviral Agents Oct. 10, 2002 WO 01/78648 6-Methylnicotinamide Derivatives As Antiviral Agents Oct. 25, 2001 WO 01/12214 MYCOPHENOLATE MOFETIL IN ASSOCIATION WITH PEG-IFN-.Alpha. Feb. 22, 2001 WO 02/100415 4′-Substituted Nucleosides Dec. 19, 2002 WO 02/18404 Nucleoside Derivatives Mar. 7, 2002 WO 02/94289 Antiviral Nucleoside Derivatives Nov. 28, 2002 WO 96/39500 Oligonucleotides Specific For Hepatitis C Virus Dec. 12, 1996 WO 03/00713 Nucleoside Compounds In Hcv Jan. 3, 2003 WO 01/60381 Nucleoside Analogs With Carboxamidine-Modified Bicyclic Base Aug. 23, 2001 WO 02/03997 Pyrido[2,3-D]Pyrimidine And Pyrimido[4,5-D]Pyrimidine Nucleosides Jan. 17, 2002 WO 97/26883 Modulation Of Th1/Th2 Cytokine Expression By Ribavirin3 And Ribavirin3 Analogs In Jul. 31, 1997 Activated T-Lymphocytes WO 03/26589 Methods And Compositions For Treating Hepatitis C Virus Using 4′-Modified Nucleosides Apr. 3, 2003 WO 03/26675 Methods And Compositions For Treating Flaviviruses And Pestiviruses Using 4′-Modified Apr. 3, 2003 Nucleoside WO 97/30067 Sugar-Modified Gapped Oligonucleotides Aug. 21, 1997 WO 01/47883 Fused-Ring Compounds And Use Thereof As Drugs Jul. 5, 2001 WO 03/00254 Fused Cyclic Compounds And Medicinal Use Thereof Jan. 3, 2003 WO 02/100354 Pyrrolo[2,3-D]Pyrimidine Nucleoside Analogs Dec. 19, 2002 WO 01/55111 Biaryl Compounds, Their Preparation And Their Use In Therapy Aug. 2, 2001 WO 01/16149 2-Azapurine Compounds And Their Use Mar. 8, 2001 WO 01/85770 Sentinel Virus Ii Nov. 15, 2001 WO 02/12263 Nucleic Acid Binding Compounds Containing Pyrazolo[3,4-D]Pyrimidine Analogues Of Purin- Feb. 14, 2002 2,6-Diamine And Their Uses JP 2001-247550 A2 Condensed Ring Compound And Its Medicinal Use Sep. 11, 2001 6210675 PT-NANB Hepatitis Polypeptides Apr. 3, 2001 6451991 Sugar-Modified Gapped Oligonucleotides Sep. 17, 2002 5830455 Method Of Treatment Using A Therapeutic Combination Of α-Interferon And Free Radical Nov. 3, 1998 Scavengers 5908621 Polyethylene Glycol Modified Interferon Therapy Jun. 1, 1999 5990276 Synthetic Inhibitors Of Hepatitis C Virus NS3 Protease Nov. 23, 1999 6172046 Combination Therapy For Eradicating Detectable HCV-RNA In Patients Having Chronic Jan. 9, 2001 Hepatitis C Infection 6177074 Polyethylene Glycol Modified Interferon Therapy Jan. 23, 2001 6326137 Hepatitis C Virus Protease-Dependent Chimeric Pestivirus Dec. 4, 2001 6434489 Compositions Of Hepatitis C Virus NS5B Polymerase And Methods For Crystallizing Same Aug. 13, 2002 6461605 Continuous Low-Dose Cytokine Infusion Therapy Oct. 8, 2002 6472373 Combination Therapy For Eradicating Detectable HCV-RNA In Antiviral Treatment Naive Oct. 29, 2002 Patients Having Chronic Hepatitis C Infection 6524570 Polyethylene Glycol Modified Interferon Therapy Feb. 25, 2003 WO 00/37097 Ribavirin-Interferon Alfa Induction Hcv Combination Therapy Jun. 29, 2000 WO 00/37110 Ribavirin-Pegylated Interferon Alfa Induction Hcv Combination Therapy Jun. 29, 2000 WO 00/62799 Hcv Combination Therapy, Containing Ribavirin In Association With Antioxidants Oct. 26, 2000 WO 01/58929 Azapeptides Useful In The Treatment Of Hepatitis C Aug. 16, 2001 WO 02/32414 Ribavirin-Pegylated Interferon Alfa Hcv Combination Therapy Apr. 25, 2002 WO 03/24461 Hcv Combination Therapy Mar. 27, 2003 WO 93/20835 Treatment Of Hepatitis With Gm-Csf Oct. 28, 1993 WO 96/36702 Soluble, Active Hepatitis C Virus Protease Nov. 21, 1996 WO 97/16204 Continuous Low-Dose Cytokine Infusion Therapy May 9, 1997 WO 97/43310 Synthetic Inhibitors Of Hepatitis C Virus Ns3 Protease Nov. 20, 1997 WO 98/48840 Polyethylene Glycol-Interferon Alpha Conjugates For Therapy Of Infection Nov. 5, 1998 WO 99/15194 Combination Therapy For Eradicating Detectable Hcv-Rna In Patients Having Chronic Apr. 1, 1999 Hepatitis C Infection WO 99/59621 Combination Therapy Comprising Ribavirin And Interferon Alpha In Antiviral Treatment Nov. 25, 1999 Naive Patients Having G Chronic Hepatitis C Infection WO 02/100846 Compounds And Methods For The Treatment Or Prevention Of Flavivirus Infections Dec. 19, 2002 WO 02/100851 Compounds And Methods For The Treatment Or Prevention Of Flavivirus Infections Dec. 19, 2002 5241053 Fused Proteins Comprising Glycoprotein Gd Of HSV-1 And LTB Aug. 31, 1993 5556946 Interleukin-2/Viral Antigen Protein Chimers Sep. 17, 1996 6087484 Enhancement Of Ribozyme Catalytic Activity By A 2′-O-Substituted Facilitator Jul. 11, 2000 Oligonucleotide 5830905 Compounds, Compositions And Methods For Treatment Of Hepatitis C Nov. 3, 1998 6316492 Methods For Treating Or Preventing Viral Infections And Associated Diseases Nov. 13, 2001 6440985 Methods For Treating Viral Infections Aug. 27, 2002 WO 00/10573 Compounds, Compositions And Methods For Treating Or Preventing Viral Infections And Mar. 2, 2000 Associated Diseases WO 00/13708 Methods For Treating Or Preventing Viral Infections And Associated Diseases Mar. 16, 2000 WO 00/18231 Methods For Treating Or Preventing Viral Infections And Associated Diseases Apr. 6, 2000 WO 99/51781 Hepatitis C Virus Ns5b Compositions And Methods Of Use Thereof Oct. 14, 1999 6323180 Hepatitis C Inhibitor Tri-Peptides Nov. 27, 2001 6143715 Hepatitis C Inhibitor Peptide Analogues Nov. 7, 2000 6329379 Hepatitis C Inhibitor Tri-Peptides Dec. 11, 2001 6329417 Hepatitis C Inhibitor Tri-Pepitides Dec. 11, 2001 6410531 Hepatitis C Inhibitor Tri-Peptides Jun. 25, 2002 6420380 Hepatitis C Inhibitor Tri-Peptides Jul. 16, 2002 6448281 Viral Polymerase Inhibitors Sep. 10, 2002 6479508 Viral Polymerase Inhibitors Nov. 12, 2002 6534523 Hepatitis C Inhibitor Tri-Peptides Mar. 18, 2003 WO 00/09543 Hepatitis C Inhibitor Tri-Peptides Feb. 24, 2000 WO 00/09558 Hepatitis C Inhibitor Peptides Feb. 24, 2000 WO 00/59929 Macrocyclic Peptides Active Against The Hepatitis C Virus Oct. 12, 2000 WO 02/04425 Viral Polymerase Inhibitors Jan. 17, 2002 WO 02/70739 Hcv Polymerase Inhibitor Assay Sep. 12, 2002 WO 03/07945 Viral Polymerase Inhibitors Jan. 30, 2003 WO 03/10140 Viral Polymerase Inhibitors Feb. 6, 2003 WO 03/10141 Viral Polymerase Inhibitors Feb. 6, 2003 WO 99/07734 Hepatitis C Inhibitor Peptide Analogues Feb. 18, 1999 WO 01/16379 Hepatitis C Virus Replication Inhibitors Mar. 8, 2001 WO 02/07761 Inhibiting Hepatitis C Virus Processing And Replication Jan. 31, 2002 WO 02/57287 Nucleoside Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase Jul. 25, 2002 WO 02/57425 Nucleoside Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase Jul. 25, 2002 WO 02/70651 Viral Reporter Particles Sep. 12, 2002 WO 03/20222 Dioxolane And Oxathiolane Derivatives As Inhibitors Of Rna-Dependent Rna Viral Polymerase Mar. 13, 2003 PCT/US2003/ Thiosemicarbazones as Anti-Virals and Immunopotentiators Jan. 10, 2003 041493

TABLE 3 US Patents and published international patent applications relating to inhalation technology for the delivery of antiviral compounds of the invention. Publication Publication Number Title Date 5740794 Apparatus and methods for dispersing dry powder medicaments Apr. 21, 1998 5775320 Method and device for delivering aerosolized medicaments Jul. 7, 1998 5785049 Method and apparatus for dispersion of dry powder medicaments Jul. 28, 1998 5814607 Pulmonary delivery of active fragments of parathyroid hormone Sep. 29, 1998 5826633 Powder filling systems, apparatus and methods Oct. 27, 1998 5458135 Method and device for delivering aerosolized medicaments Oct. 17, 1995 5607915 Pulmonary delivery of active fragments of parathyroid hormone Mar. 4, 1997 5654007 Methods and system for processing dispersible fine powders Aug. 5, 1997 5922354 Methods and system for processing dispersible fine powders Jul. 13, 1999 5928469 Process for storage of materials Jul. 27, 1999 5976574 Processes for spray drying hydrophobic drugs in organic solvent suspensions Nov. 2, 1999 5985248 processes for spray drying solutions of hydrophobic drugs and compositions thereof Nov. 16, 1999 5994314 Compositions and methods for nucleic acid delivery to the lung Nov. 30, 1999 5997848 Methods and compositions for pulmonary delivery of insulin Dec. 7, 1999 6001336 Processes for spray drying aqueous suspensions of hydrophobic drugs and compositions thereof Dec. 14, 1999 6019968 Dispersible antibody compositions and methods for their preparation and use Feb. 1, 2000 6051256 Dispersible macromolecule compositions and methods for their preparation and use Apr. 18, 2000 6071428 Stable compositions Jun. 6, 2000 6077543 Systems and processes for spray drying hydrophobic drugs with hydrophilic excipients Jun. 20, 2000 6080721 Pulmonary delivery of active fragments of parathyroid hormone Jun. 27, 2000 6089228 Apparatus and methods for dispersing dry powder medicaments Jul. 18, 2000 6103270 Methods and system for processing dispersible fine powders Aug. 15, 2000 6123936 Methods and compositions for the dry powder formulation of interferons Sep. 26, 2000 6136346 Powdered pharmaceutical formulations having improved dispersibility Oct. 24, 2000 6138668 Method and device for delivering aerosolized medicaments Oct. 31, 2000 6165463 Dispersible antibody compositions and methods for their preparation and use Dec. 26, 2000 6182712 Power filling apparatus and methods for their use Feb. 6, 2001 6187344 Powdered pharmaceutical formulations having improved dispersibility Feb. 13, 2001 6207135 Gaseous microparticles for ultrasonic diagnosis and process for their production Mar. 27, 2001 6231851 Methods and compositions for the dry powder formulation of interferons May 15, 2001 6257233 Dry powder dispersing apparatus and methods for their use Jul. 10, 2001 6258341 Stable glassy state powder formulations Jul. 10, 2001 6267155 Powder filling systems, apparatus and methods Jul. 31, 2001 6294204 Method of producing morphologically uniform microcapsules and microcapsules produced by Sep. 25, 2001 this method 6303582 Compositions and methods for nucleic acid delivery to the lung Oct. 16, 2001 6309623 Stabilized preparations for use in metered dose inhalers Oct. 30, 2001 6309671 Stable glassy state powder formulations Oct. 30, 2001 6358530 Powdered pharmaceutical formulations having improved dispersibility Mar. 19, 2002 6365190 Systems and processes for spray drying hydrophobic drugs with hydrophilic excipients Apr. 2, 2002 6372258 Methods of spray-drying a drug and a hydrophobic amino acid Apr. 16, 2002 6423344 Dispersible macromolecule compositions and methods for their preparation and use Jul. 23, 2002 6426210 Storage of materials Jul. 30, 2002 6433040 Stabilized bioactive preparations and methods of use Aug. 13, 2002 6440337 Method and apparatus for the formation of particles Aug. 27, 2002 RE37872 Storage of materials Oct. 8, 2002 6479049 Methods and compositions for the dry powder formulation of interferons Nov. 12, 2002 6503411 Stable compositions Jan. 7, 2003 6509006 Devices compositions and methods for the pulmonary delivery of aerosolized medicaments Jan. 21, 2003 6514496 Dispersible antibody compositions and methods for their preparation and use Feb. 4, 2003 6518239 dry powder compositions having improved dispersivity Feb. 11, 2003 6543448 apparatus and methods for dispersing dry powder medicaments Apr. 8, 2003 6546929 dry powder dispersing apparatus and methods for their use Apr. 15, 2003 WO 00/15262 dry powder active agent pulmonary delivery Mar. 23, 2000 WO 93/00951 method and device for delivering aerosolized medicaments Jan. 21, 1993 WO 94/07514 pulmonary delivery of active fragments of parathyroid hormone Apr. 14, 1994 WO 95/24183 methods and compositions for pulmonary delivery of insulin Sep. 14, 1995 WO 95/31479 methods and compositions for the dry powder formulation of interferons Nov. 23, 1995 WO 96/09085 apparatus and methods for dispersing dry powder medicaments Mar. 28, 1996 WO 96/32096 powdered pharmaceutical formulations having improved dispersibility Oct. 17, 1996 WO 96/32116 compositions and methods for nucleic acid delivery to the lung Oct. 17, 1996 WO 96/32149 pulmonary delivery of aerosolized medicaments Oct. 17, 1996 WO 96/32152 pulmonary administration of dry powder alpha 1-antitrypsin Oct. 17, 1996 WO 96/40068 methods and system for processing dispersible fine powders Dec. 19, 1996 WO 97/41031 powder filling systems, apparatus and methods Nov. 6, 1997 WO 97/41833 dispersible macromolecule compositions and methods for their preparation and use Nov. 13, 1997 WO 98/16205 stable glassy state powder formulations Apr. 23, 1998 WO 98/29096 aerosolized hydrophobic drug Jul. 9, 1998 WO 98/29098 processes for spray drying aqueous suspensions of hydrophobic drugs with hydrophilic Jul. 9, 1998 excipients and compositions prepared by such processes WO 98/29140 processes and compositions for spray drying hydrophobic drugs in organic solvent suspensions Jul. 9, 1998 of hydrophilic excipients WO 98/29141 processes for spray drying solutions of hydrophobic drugs with hydrophilic excipients and Jul. 9, 1998 compositions prepared by such processes WO 99/19215 powder filling apparatus and method Apr. 22, 1999 WO 99/42124 liquid crystal forms of cyclosporin Aug. 26, 1999 WO 99/47196 aerosolized active agent delivery Sep. 23, 1999 WO 99/62495 dry powder dispersing apparatus and methods for their use Dec. 9, 1999 WO 00/21594 flow resistance modulated aerosolized active agent delivery Apr. 20, 2000 WO 00/61178 pulmonary administration of dry powder formulations for treating infertility Oct. 19, 2000 WO 00/72904 apparatus and method for dispensing metered amount of aerosolized medication Dec. 7, 2000 WO 01/00263 systems and methods for aerosolizing pharmaceutical formulations Jan. 4, 2001 WO 01/00312 spray drying process for preparing dry powders Jan. 4, 2001 WO 01/32144 dry powder compositions having improved dispersivity May 10, 2001 WO 01/43529 receptacles to facilitate the extraction of powders Jun. 21, 2001 WO 01/43530 systems and methods for extracting powders from receptacles Jun. 21, 2001 WO 01/43802 systems and methods for treating packaged powders Jun. 21, 2001 WO 01/44764 systems and methods for non-destructive mass sensing Jun. 21, 2001 WO 01/87393 systems, devices and methods for opening receptacles having a powder to be fluidized Nov. 22, 2001 WO 01/93932 lockout mechanism for aerosol drug delivery devices Dec. 13, 2001 WO 02/09669 apparatus and process to produce particles having a narrow size distribution and particles made Feb. 7, 2002 thereby WO 02/11695 inhaleable spray dried 4-helix bundle protein powders having minimized aggregation Feb. 14, 2002 WO 02/49619 induced phase transition method for the production of microparticles containing hydrophilic Jun. 27, 2002 active agents WO 02/49620 induced phase transition method for the production of microparticles containing hydrophobic Jun. 27, 2002 active agents WO 02/54868 pulmonary delivery of polyene antifungal agents Jul. 18, 2002 WO 02/87542 novel methods and compositions for delivering macromolecules to or via the respiratory tract Nov. 7, 2002 WO 02/100548 centrifuged rotating drum for treating cohesive powders Dec. 19, 2002 WO 03/00326 powder aerosolization apparatus and method Jan. 3, 2003 WO 03/00329 flow regulator for aerosol drug delivery device and methods Jan. 3, 2003

TABLE 4 Forward and reverse primers for nucleic acid amplification of SARSV For- Forward ward Reverse Prim- Prod- Opti- Pair primer Forward Forward Prim- Forward primer Reverse Reverse Reverse Reverse er Prod- uct An- mum Numb- SEQ Primer Primer er Primer SEQ Primer Primer Primer Primer Tm Product uct % neal Anneal er ID NO Start Stop Tm % GC ID NO Start Stop Tm % GC Diff Length Tm GC Score Temp 1 1021 12726 12746 51.3 47.6 3521 12996 12977 50.2 40 1 271 75 42.8 26 52.6 2 1022 12236 12256 51.2 42.9 3522 12993 12975 51.4 47.4 0.2 758 76.4 42.5 26 54 3 1023 12373 12391 50.8 47.4 3523 12993 12975 51.4 47.4 0.6 621 76.4 43 26 53.8 4 1024 12236 12256 51.2 42.9 3524 12996 12977 50.2 40 0.9 761 76.4 42.3 26 53.6 5 1025 12373 12391 50.8 47.4 3525 12996 12977 50.2 40 0.5 624 76.4 42.8 26 53.6 6 1026 12726 12746 51.3 47.6 3526 12993 12975 51.4 47.4 0.1 268 75.1 43.3 26 53.1 7 1027 2671 2692 52.1 40.9 3527 3185 3164 51 45.5 1.2 515 75.6 41.6 26 53.3 8 1028 28942 28961 50.2 45 3528 29298 29280 51.4 52.6 1.2 357 76.4 44.8 26 53.6 9 1029 19801 19819 53.2 52.6 3529 19922 19901 51.5 45.5 1.7 122 72.2 43.4 26 51.1 10 1030 19800 19817 50.4 50 3530 19921 19901 50.2 47.6 0.3 122 72.2 43.4 26 50.7 11 1031 9930 9948 51.5 52.6 3531 10605 10588 51.1 50 0.4 676 75.8 41.3 27 53.5 12 1032 9933 9952 50.9 45 3532 10605 10588 51.1 50 0.2 673 75.8 41.2 27 53.4 13 1033 9930 9949 52.2 50 3533 10605 10588 51.1 50 1.1 676 75.8 41.3 27 53.5 14 1034 9927 9945 50.8 52.6 3534 10605 10588 51.1 50 0.3 679 75.8 41.2 28 53.4 15 1035 3789 3806 50 50 3535 4445 4425 50.6 42.9 0.5 657 75.5 40.5 28 52.9 16 1036 3788 3805 50 50 3536 4444 4424 50.6 42.9 0.5 657 75.5 40.5 28 52.9 17 1037 3795 3813 52.1 52.6 3537 4445 4425 50.6 42.9 1.5 651 75.5 40.6 28 53.1 18 1038 3787 3804 50 50 3538 4445 4425 50.6 42.9 0.5 659 75.4 40.4 28 52.9 19 1039 19801 19819 53.2 52.6 3539 19921 19900 51.8 45.5 1.4 121 72.3 43.8 28 51.2 20 1040 24418 24436 50 47.4 3540 25182 25164 51.4 47.4 1.4 765 76.1 41.7 28 53.4 21 1041 9929 9949 53.8 47.6 3541 10449 10425 54.6 40 0.8 521 75.4 40.9 28 54 22 1042 2671 2692 52.1 40.9 3542 3186 3165 50.4 40.9 1.7 516 75.6 41.5 28 53.1 23 1043 3792 3810 52.9 52.6 3543 4446 4425 51.8 45.5 1.1 655 75.5 40.6 28 53.5 24 1044 9933 9952 50.9 45 3544 10449 10431 50.9 47.4 0.1 517 75.3 40.8 28 53.1 25 1045 3792 3810 52.9 52.6 3545 4445 4424 51.3 40.9 1.6 654 75.5 40.5 28 53.3 26 1046 25782 25806 53.5 40 3546 26184 26164 52.4 42.9 1.1 403 74.7 40.2 28 53.1 27 1047 9927 9945 50.8 52.6 3547 10449 10431 50.9 47.4 0.1 523 75.4 40.9 28 53.1 28 1048 9927 9945 50.8 52.6 3548 10449 10428 51.9 40.9 1.1 523 75.4 40.9 28 53.1 29 1049 3789 3806 50 50 3549 4444 4424 50.6 42.9 0.5 656 75.5 40.5 28 53 30 1050 3795 3813 52.1 52.6 3550 4444 4424 50.6 42.9 1.5 650 75.5 40.6 28 53.1 31 1051 9933 9952 50.9 45 3551 10449 10428 51.9 40.9 1.1 517 75.3 40.8 28 53.1 32 1052 9930 9948 51.5 52.6 3552 10449 10431 50.9 47.4 0.5 520 75.4 41 28 53.2 33 1053 9930 9948 51.5 52.6 3553 10449 10428 51.9 40.9 0.4 520 75.4 41 28 53.3 34 1054 9929 9948 53.2 50 3554 10449 10425 54.6 40 1.4 521 75.4 40.9 28 53.8 35 1055 9931 9952 53 45.5 3555 10449 10425 54.6 40 1.6 519 75.3 40.8 28 53.7 36 1056 3791 3808 50 50 3556 4445 4425 50.6 42.9 0.5 655 75.5 40.5 28 52.9 37 1057 3791 3808 50 50 3557 4444 4424 50.6 42.9 0.5 654 75.5 40.5 28 53 38 1058 9930 9949 52.2 50 3558 10449 10431 50.9 47.4 1.2 520 75.4 41 28 53.2 39 1059 9930 9949 52.2 50 3559 10449 10428 51.9 40.9 0.3 520 75.4 41 28 53.5 40 1060 3788 3805 50 50 3560 4445 4425 50.6 42.9 0.5 658 75.5 40.4 28 52.9 41 1061 19800 19817 50.4 50 3561 19921 19900 51.8 45.5 1.4 122 72.2 43.4 28 50.8 42 1062 3787 3804 50 50 3562 4444 4424 50.6 42.9 0.5 658 75.5 40.4 28 52.9 43 1063 25782 25806 53.5 40 3563 26183 26163 51.7 42.9 1.7 402 74.7 40.3 28 52.9 44 1064 25782 25806 53.5 40 3564 26183 26160 54.5 41.7 1 402 74.7 40.3 28 53.5 45 1065 25782 25806 53.5 40 3565 26183 26159 54.9 40 1.5 402 74.7 40.3 28 53.5 46 1066 2429 2447 50.2 47.4 3566 3187 3166 50.3 45.5 0.1 759 76.6 43 29 53.8 47 1067 2427 2445 52.1 52.6 3567 3185 3164 51 45.5 1.1 759 76.7 43.1 29 54.1 48 1068 2429 2447 50.2 47.4 3568 3185 3164 51 45.5 0.7 757 76.6 42.9 29 53.8 49 1069 19800 19817 50.4 50 3569 19923 19904 50.1 50 0.3 124 72.3 43.5 29 50.8 50 1070 2427 2445 52.1 52.6 3570 3187 3166 50.3 45.5 1.8 761 76.7 43.1 29 53.9 51 1071 29183 29204 50.4 40.9 3571 29412 29393 50.3 45 0 230 75.3 44.8 29 52.9 52 1072 16367 16386 51.4 50 3572 16780 16760 51.4 42.9 0.1 414 75 40.8 30 53 53 1073 11543 11562 50.4 40 3573 12254 12236 50.5 47.4 0.1 712 76.2 42 30 53.6 54 1074 12976 12995 51.1 45 3574 13547 13528 50.2 45 0.9 572 77.4 45.5 30 54.3 55 1075 12040 12057 50.6 50 3575 12254 12236 50.5 47.4 0.1 215 75.5 45.6 30 53.1 56 1076 12976 12996 51.8 42.9 3576 13544 13525 52.6 55 0.8 569 77.5 45.7 30 54.8 57 1077 10141 10160 51 45 3577 10605 10588 51.1 50 0.1 465 74.9 40.2 30 52.8 58 1078 12235 12253 50.1 52.6 3578 12996 12977 50.2 40 0.1 762 76.4 42.4 30 53.6 59 1079 19795 19814 50.4 45 3579 19921 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28672 28654 50.6 52.6 0.5 279 78.6 51.6 30 55.3 85 1105 28395 28413 50.2 42.1 3605 28672 28654 50.6 52.6 0.4 278 78.6 51.4 30 55.2 86 1106 28396 28415 51.2 45 3606 28672 28654 50.6 52.6 0.6 277 78.6 51.6 30 55.3 87 1107 26421 26441 51.5 42.9 3607 26587 26568 52.7 45 1.2 167 72.3 40.1 30 51.2 88 1108 26421 26441 51.5 42.9 3608 26589 26571 51.7 47.4 0.2 169 72.4 40.2 30 51.2 89 1109 26421 26441 51.5 42.9 3609 26589 26572 51 50 0.5 169 72.4 40.2 30 51.1 90 1110 26421 26441 51.5 42.9 3610 26590 26573 51.7 50 0.3 170 72.3 40 30 51.2 91 1111 26040 26061 56.4 54.5 3611 26589 26568 55.2 45.5 1.2 550 75.1 40 30 54.2 92 1112 26039 26057 52.6 52.6 3612 26183 26160 54.5 41.7 1.9 145 71.9 40.7 30 51.2 93 1113 26039 26057 52.6 52.6 3613 26182 26161 51.2 40.9 1.4 144 71.7 40.3 30 50.7 94 1114 26039 26057 52.6 52.6 3614 26183 26163 51.7 42.9 0.9 145 71.9 40.7 30 51 95 1115 8867 8887 52.3 47.6 3615 9253 9235 51.6 47.4 0.7 387 75.1 41.3 30 53.2 96 1116 10247 10267 50.5 47.6 3616 10605 10588 51.1 50 0.6 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25182 25164 51.4 47.4 1.5 765 76.1 41.7 32 53.8 135 1155 24420 24440 50.8 42.9 3655 25182 25164 51.4 47.4 0.6 763 76.1 41.5 32 53.6 136 1156 8867 8887 52.3 47.6 3656 9107 9086 51.6 45.5 0.7 241 74.1 41.5 32 52.5 137 1157 1402 1422 50.2 42.9 3657 2103 2083 50.6 42.9 0.4 702 76.7 43.3 32 53.8 138 1158 25782 25805 52.1 41.7 3658 26183 26163 51.7 42.9 0.4 402 74.7 40.3 32 52.9 139 1159 25781 25805 53.5 40 3659 26183 26160 54.5 41.7 1 403 74.7 40.2 32 53.4 140 1160 25781 25805 53.5 40 3660 26183 26159 54.9 40 1.5 403 74.7 40.2 32 53.4 141 1161 2671 2692 52.1 40.9 3661 3052 3033 50.3 50 1.8 382 74.8 40.6 32 52.5 142 1162 12726 12746 51.3 47.6 3662 13177 13156 50.4 40.9 0.9 452 76.4 43.8 32 53.7 143 1163 16909 16928 50.8 45 3663 17111 17090 51.1 40.9 0.3 203 75 44.8 32 52.8 144 1164 12234 12252 50.6 47.4 3664 12993 12975 51.4 47.4 0.8 760 76.4 42.5 32 53.8 145 1165 26039 26057 52.6 52.6 3665 26828 26810 52.9 52.6 0.2 790 76.4 42.4 32 54.4 146 1166 26039 26057 52.6 52.6 3666 26694 26677 51.4 50 1.2 656 75.7 41 32 53.5 147 1167 26039 26057 52.6 52.6 3667 26692 26674 51.9 52.6 0.7 654 75.7 41 32 53.6 148 1168 26039 26057 52.6 52.6 3668 26691 26673 51.3 47.4 1.3 653 75.6 40.9 32 53.4 149 1169 26039 26057 52.6 52.6 3669 26687 26669 51.3 47.4 1.3 649 75.6 40.8 32 53.4 150 1170 26039 26057 52.6 52.6 3670 26684 26666 53.4 52.6 0.8 646 75.6 40.9 32 53.8 151 1171 26039 26057 52.6 52.6 3671 26683 26665 52.7 52.6 0.1 645 75.6 40.9 32 53.8 152 1172 9934 9953 50.7 50 3672 10449 10431 50.9 47.4 0.2 516 75.4 40.9 32 53.1 153 1173 9927 9945 50.8 52.6 3673 10455 10434 51.1 40.9 0.3 529 75.3 40.6 32 53 154 1174 7728 7746 51.7 52.6 3674 8188 8169 50.5 45 1.2 461 75.6 41.9 32 53.2 155 1175 18550 18571 50.4 40.9 3675 19216 19195 50.2 40.9 0.2 667 75.7 41.1 32 53.2 156 1176 19801 19819 53.2 52.6 3676 19921 19899 52.4 43.5 0.8 121 72.3 43.8 32 51.4 157 1177 19709 19730 51.3 40.9 3677 19923 19904 50.1 50 1.2 215 73.9 41.9 32 51.9 158 1178 4639 4659 51.1 47.6 3678 5306 5289 50.8 50 0.3 668 75.6 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440 76.9 45.2 32 55 184 1204 28867 28887 53.7 47.6 3704 29301 29282 55.3 55 1.5 435 76.9 45.3 32 55.1 185 1205 28868 28888 51.4 42.9 3705 29298 29280 51.4 52.6 0 431 76.8 45 32 54.3 186 1206 28867 28888 54.3 45.5 3706 29306 29288 53.5 52.6 0.8 440 76.9 45.2 32 55 187 1207 28867 28888 54.3 45.5 3707 29301 29282 55.3 55 1 435 76.9 45.3 32 55.2 188 1208 28870 28889 50.1 40 3708 29298 29280 51.4 52.6 1.3 429 76.8 45 32 53.9 189 1209 28868 28889 52 40.9 3709 29306 29288 53.5 52.6 1.5 439 76.9 45.1 32 54.5 190 1210 28867 28889 54.8 43.5 3710 29301 29282 55.3 55 0.5 435 76.9 45.3 32 55.4 191 1211 28867 28890 55.2 41.7 3711 29306 29288 53.5 52.6 1.7 440 76.9 45.2 32 55 192 1212 28867 28890 55.2 41.7 3712 29301 29282 55.3 55 0.1 435 76.9 45.3 32 55.5 193 1213 28867 28890 55.2 41.7 3713 29299 29280 53.9 55 1.3 433 76.9 45.3 32 55.1 194 1214 12234 12252 50.6 47.4 3714 12996 12977 50.2 40 0.3 763 76.4 42.3 32 53.6 195 1215 28968 28988 50.9 47.6 3715 29298 29280 51.4 52.6 0.6 331 76.2 44.7 32 53.7 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29273 53.1 50 3728 29358 29339 52.8 50 0.2 105 73.4 48.6 32 52.3 209 1229 29259 29278 52.6 50 3729 29358 29339 52.8 50 0.2 100 72.4 47 32 51.6 210 1230 1402 1422 50.2 42.9 3730 1773 1755 51.7 52.6 1.5 372 75.8 43.3 33 53.2 211 1231 12726 12746 51.3 47.6 3731 13326 13306 50.7 42.9 0.6 601 76.7 43.6 33 54 212 1232 4 22 52.3 52.6 3732 269 251 51.1 52.6 1.2 266 76.5 46.6 33 54 213 1233 19800 19817 50.4 50 3733 19923 19903 50.9 47.6 0.4 124 72.3 43.5 33 50.9 214 1234 2371 2389 50.3 47.4 3734 3082 3058 52.3 40 2 712 76.7 43.3 33 53.9 215 1235 3 21 53.4 52.6 3735 270 251 52.9 50 0.5 268 76.4 46.3 33 54.4 216 1236 9930 9949 52.2 50 3736 10183 10166 50.9 50 1.3 254 75.3 44.1 33 53.1 217 1237 19795 19814 50.4 45 3737 19923 19904 50.1 50 0.3 129 72.5 43.4 33 50.9 218 1238 8867 8887 52.3 47.6 3738 9365 9347 53 52.6 0.7 499 75.8 42.1 33 53.9 219 1239 2371 2389 50.3 47.4 3739 3055 3036 50.6 50 0.3 685 76.7 43.4 33 53.9 220 1240 19709 19730 51.3 40.9 3740 19921 19901 50.2 47.6 1.1 213 73.9 41.8 33 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52 52.6 0.5 182 73.8 42.9 34 52.3 358 1378 26421 26441 51.5 42.9 3878 26655 26634 50.6 40.9 0.9 235 74.1 41.7 34 52.2 359 1379 26421 26441 51.5 42.9 3879 26657 26639 50.8 47.4 0.7 237 74.2 41.8 34 52.3 360 1380 26040 26061 56.4 54.5 3880 26183 26159 54.9 40 1.5 144 72 41 34 52 361 1381 26040 26061 56.4 54.5 3881 26183 26160 54.5 41.7 2 144 72 41 34 51.9 362 1382 26040 26061 56.4 54.5 3882 26184 26161 55.1 41.7 1.3 145 71.9 40.7 34 52 363 1383 12373 12391 50.8 47.4 3883 12724 12705 52.4 55 1.6 352 75.6 42.9 34 53.2 364 1384 26040 26061 56.4 54.5 3884 26589 26569 54.7 47.6 1.7 550 75.1 40 34 54.1 365 1385 26039 26058 54 55 3885 26183 26159 54.9 40 0.9 145 71.9 40.7 34 51.7 366 1386 26039 26058 54 55 3886 26183 26160 54.5 41.7 0.4 145 71.9 40.7 34 51.7 367 1387 26039 26058 54 55 3887 26183 26161 54 43.5 0 145 71.9 40.7 34 51.7 368 1388 26039 26058 54 55 3888 26184 26163 53 40.9 1 146 71.8 40.4 34 51.3 369 1389 26039 26057 52.6 52.6 3889 26174 26153 51 40.9 1.6 136 71.8 41.2 34 50.7 370 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624 76.4 42.9 35 53.6 596 1616 8374 8393 51.2 45 4116 8928 8911 51.9 50 0.7 555 75.1 40 35 53 597 1617 24174 24194 50.9 42.9 4117 24936 24919 51.8 50 0.8 763 75.8 41 35 53.5 598 1618 24179 24198 51 45 4118 24936 24919 51.8 50 0.7 758 75.8 41 35 53.5 599 1619 7679 7698 50.6 50 4119 8049 8032 50.4 50 0.2 371 75.4 42.3 35 53 600 1620 13177 13197 50.3 42.9 4120 13320 13300 51.4 47.6 1.1 144 73.2 43.8 35 51.4 601 1621 24179 24200 53.3 40.9 4121 24934 24913 53.4 45.5 0.2 756 75.8 41 35 54.2 602 1622 9927 9945 50.8 52.6 4122 10670 10649 51.3 40.9 0.5 744 75.7 40.9 35 53.4 603 1623 2427 2445 52.1 52.6 4123 3052 3033 50.3 50 1.8 626 76.4 42.8 35 53.6 604 1624 24418 24436 50 47.4 4124 24527 24507 51 42.9 1 110 71.3 42.7 35 50 605 1625 24417 24436 52.6 50 4125 24517 24494 53.2 41.7 0.6 101 71.1 43.6 35 50.6 606 1626 8375 8396 51.8 45.5 4126 8929 8911 53.4 52.6 1.6 555 75.1 40 35 53.2 607 1627 24418 24439 52.9 45.5 4127 25080 25062 53.5 52.6 0.6 663 75.8 41.2 35 54 608 1628 18074 18094 51.1 42.9 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798 1818 28968 28988 50.9 47.6 4318 29358 29339 52.8 50 2 391 76.4 44.5 36 53.9 799 1819 11545 11563 50.8 47.4 4319 12257 12237 51.3 47.6 0.5 713 76.2 42.1 36 53.7 800 1820 24417 24436 52.6 50 4320 25080 25062 53.5 52.6 0.9 664 75.8 41.3 36 53.9 801 1821 28968 28989 51.5 45.5 4321 29358 29339 52.8 50 1.3 391 76.4 44.5 36 54.1 802 1822 3789 3808 53.5 50 4322 4318 4294 54.4 40 0.9 530 75.5 41.1 36 54 803 1823 3232 3252 51.1 47.6 4323 3646 3625 52 40.9 0.9 415 75.3 41.4 36 53.1 804 1824 3232 3252 51.1 47.6 4324 3647 3628 50.6 45 0.5 416 75.3 41.6 36 53 805 1825 28971 28993 51.9 43.5 4325 29306 29288 53.5 52.6 1.6 336 76.2 44.6 36 54 806 1826 24179 24200 53.3 40.9 4326 24818 24797 51.6 40.9 1.6 640 75.8 41.2 36 53.6 807 1827 3231 3251 52 47.6 4327 3650 3631 53.1 50 1.1 420 75.4 41.7 36 53.5 808 1828 9930 9950 52.6 47.6 4328 10449 10425 54.6 40 2 520 75.4 41 36 53.7 809 1829 8866 8885 51.1 45 4329 9254 9236 50.6 47.4 0.5 389 75 41.1 36 52.8 810 1830 2522 2541 51.4 45 4330 2672 2653 51.6 50 0.2 151 75.3 48.3 36 53.2 811 1831 11541 11561 50.9 42.9 4331 12258 12238 50.3 42.9 0.6 718 76.2 41.9 36 53.5 812 1832 3232 3251 50.3 50 4332 3646 3625 52 40.9 1.7 415 75.3 41.4 36 52.9 813 1833 3232 3251 50.3 50 4333 3647 3628 50.6 45 0.3 416 75.3 41.6 36 52.9 814 1834 23843 23863 50.3 42.9 4334 24527 24508 50.5 45 0.2 685 76 41.8 36 53.4 815 1835 21210 21228 53.2 52.6 4335 21317 21293 53.2 40 0 108 71.1 42.6 36 50.8 816 1836 3229 3249 51.4 47.6 4336 3650 3631 53.1 50 1.7 422 75.4 41.7 36 53.3 817 1837 3230 3249 50.1 45 4337 3494 3473 50.4 40.9 0.3 265 74.2 41.1 36 52.1 818 1838 2371 2389 50.3 47.4 4338 2997 2976 51.4 40.9 1.1 627 76.7 43.5 36 53.9 819 1839 29186 29206 51.3 42.9 4339 29298 29280 51.4 52.6 0.1 113 72.8 46 36 51.5 820 1840 9929 9946 50 50 4340 10455 10435 50.5 42.9 0.4 527 75.3 40.6 36 52.8 821 1841 9351 9370 51.2 50 4341 9989 9968 51 40.9 0.2 639 75.4 40.4 36 53.2 822 1842 25348 25365 50.4 50 4342 25772 25753 51.9 50 1.5 425 74.9 40.5 36 52.6 823 1843 1402 1422 50.2 42.9 4343 2103 2082 52 45.5 1.8 702 76.7 43.3 36 53.8 824 1844 9929 9946 50 50 4344 10608 10589 51 50 0.9 680 75.8 41.2 36 53.2 825 1845 9934 9953 50.7 50 4345 10608 10589 51 50 0.3 675 75.8 41.2 36 53.4 826 1846 13176 13196 51.4 47.6 4346 13544 13525 52.6 55 1.2 369 77.1 46.3 36 54.5 827 1847 7725 7743 50.8 47.4 4347 8189 8170 50.6 50 0.2 465 75.6 41.9 36 53.2 828 1848 7725 7743 50.8 47.4 4348 8190 8172 50.3 47.4 0.6 466 75.6 41.8 36 53.1 829 1849 18074 18093 50.3 45 4349 18662 18641 50.4 40.9 0.1 589 76.2 42.6 36 53.6 830 1850 18074 18093 50.3 45 4350 18632 18611 50.2 40.9 0.1 559 76.2 42.8 36 53.5 831 1851 29200 29222 53.2 43.5 4351 29306 29288 53.5 52.6 0.3 107 73.1 47.7 36 52.3 832 1852 25348 25366 51.2 47.4 4352 25545 25526 51.7 45 0.5 198 74.1 42.9 36 52.3 833 1853 25348 25366 51.2 47.4 4353 25545 25525 52.3 42.9 1.1 198 74.1 42.9 36 52.3 834 1854 29200 29223 53.7 41.7 4354 29306 29288 53.5 52.6 0.2 107 73.1 47.7 36 52.3 835 1855 25347 25366 52.7 50 4355 25545 25521 54.5 40 1.8 199 74.2 43.2 36 52.9 836 1856 3792 3811 54 55 4356 4447 4425 53 43.5 1 656 75.5 40.5 36 53.9 837 1857 29200 29224 54.2 40 4357 29306 29288 53.5 52.6 0.7 107 73.1 47.7 36 52.3 838 1858 985 1004 51.1 50 4358 1483 1465 50.5 47.4 0.6 499 76.4 43.5 36 53.7 839 1859 2427 2445 52.1 52.6 4359 3189 3168 51 45.5 1.1 763 76.7 43.1 36 54.1 840 1860 13701 13725 53.6 40 4360 14084 14060 53.6 40 0.1 384 74.6 40.1 36 53.4 841 1861 985 1004 51.1 50 4361 1483 1464 51.3 45 0.2 499 76.4 43.5 36 53.9 842 1862 8794 8813 51.6 45 4362 9559 9539 50.6 42.9 1 766 75.9 41.3 37 53.4 843 1863 3789 3806 50 50 4363 4435 4417 50.5 52.6 0.4 647 75.5 40.5 37 52.9 844 1864 13177 13197 50.3 42.9 4364 13314 13297 51 50 0.6 138 72.8 43.5 37 51.2 845 1865 3791 3808 50 50 4365 4435 4417 50.5 52.6 0.4 645 75.4 40.5 37 52.9 846 1866 9139 9159 52.5 47.6 4366 9364 9346 53.9 52.6 1.4 226 74.9 43.8 37 53.3 847 1867 3226 3245 51.7 55 4367 3494 3473 50.4 40.9 1.3 269 74.5 41.6 37 52.4 848 1868 13040 13059 50.9 50 4368 13314 13297 51 50 0.1 275 75.6 44.4 37 53.3 849 1869 2522 2541 51.4 45 4369 2891 2873 50.8 47.4 0.6 370 76 43.8 37 53.6 850 1870 8865 8884 50.4 45 4370 9245 9226 50 45 0.4 381 74.9 40.9 37 52.6 851 1871 3787 3804 50 50 4371 4434 4416 51.5 52.6 1.4 648 75.4 40.3 37 52.9 852 1872 3226 3245 51.7 55 4372 3646 3625 52 40.9 0.3 421 75.3 41.6 37 53.4 853 1873 3226 3245 51.7 55 4373 3647 3628 50.6 45 1.1 422 75.4 41.7 37 53.1 854 1874 3226 3245 51.7 55 4374 3650 3631 53.1 50 1.4 425 75.5 41.9 37 53.5 855 1875 2387 2405 51.6 52.6 4375 2747 2727 50 42.9 1.6 361 76.9 46 37 53.9 856 1876 18074 18093 50.3 45 4376 18229 18209 50.1 42.9 0.2 156 73.2 42.9 37 51.4 857 1877 13701 13725 53.6 40 4377 14059 14040 52.8 50 0.8 359 74.5 40.1 37 53.1 858 1878 3787 3804 50 50 4378 4435 4417 50.5 52.6 0.4 649 75.4 40.4 37 52.9 859 1879 13040 13059 50.9 50 4379 13323 13304 51.1 45 0.2 284 75.7 44.4 37 53.4 860 1880 3789 3806 50 50 4380 4434 4416 51.5 52.6 1.4 646 75.4 40.4 37 52.9 861 1881 15506 15527 50.8 40.9 4381 16214 16196 51.8 52.6 1 709 75.5 40.3 37 53.2 862 1882 12234 12252 50.6 47.4 4382 12412 12392 50 42.9 0.6 179 73.1 41.3 37 51.3 863 1883 12234 12252 50.6 47.4 4383 12739 12718 51 40.9 0.4 506 75.8 42.1 37 53.4 864 1884 18074 18094 51.1 42.9 4384 18229 18209 50.1 42.9 0.9 156 73.2 42.9 37 51.4 865 1885 18075 18095 50.6 47.6 4385 18223 18206 51.8 50 1.2 149 73.3 43.6 37 51.6 866 1886 13040 13059 50.9 50 4386 13326 13306 50.7 42.9 0.2 287 75.7 44.3 37 53.3 867 1887 18080 18098 51.2 52.6 4387 18233 18215 51.3 52.6 0.1 154 73.9 44.8 37 52.2 868 1888 18080 18098 51.2 52.6 4388 18233 18214 52 50 0.9 154 73.9 44.8 37 52.2 869 1889 18080 18098 51.2 52.6 4389 18231 18210 52.2 45.5 1 152 73.5 44.1 37 51.9 870 1890 18080 18098 51.2 52.6 4390 18223 18206 51.8 50 0.6 144 73.2 43.8 37 51.7 871 1891 18077 18098 52.9 50 4391 18220 18202 54.8 52.6 1.9 144 73.2 43.8 37 52.2 872 1892 18076 18098 54.4 47.8 4392 18443 18424 55.9 55 1.5 368 75.8 43.2 37 54.5 873 1893 3792 3810 52.9 52.6 4393 4436 4417 52.2 50 0.6 645 75.4 40.5 37 53.6 874 1894 3055 3074 51.1 50 4394 3647 3628 50.6 45 0.5 593 76.3 42.7 37 53.7 875 1895 3055 3074 51.1 50 4395 3646 3625 52 40.9 0.9 592 76.2 42.6 37 53.8 876 1896 15506 15527 50.8 40.9 4396 15645 15625 51.1 42.9 0.4 140 71.8 40.7 37 50.6 877 1897 18081 18099 51.2 52.6 4397 18229 18209 50.1 42.9 1.1 149 73.3 43.6 37 51.4 878 1898 13039 13058 51.8 50 4398 13155 13138 50.4 50 1.4 117 73.4 47 37 51.6 879 1899 3055 3075 51.8 47.6 4399 3650 3631 53.1 50 1.3 596 76.3 42.8 37 54.1 880 1900 18080 18099 53 50 4400 18223 18205 53.3 52.6 0.4 144 73.2 43.8 37 52.2 881 1901 27361 27380 52.4 55 4401 27579 27558 51.1 40.9 1.3 219 75.4 45.2 37 53.2 882 1902 3221 3239 51.5 52.6 4402 3503 3484 51.5 50 0 283 74.8 42 37 52.9 883 1903 3221 3239 51.5 52.6 4403 3504 3485 50.4 45 1.1 284 74.7 41.9 37 52.5 884 1904 18077 18099 54.4 47.8 4404 18220 18201 56.1 55 1.7 144 73.2 43.8 37 52.6 885 1905 3055 3075 51.8 47.6 4405 3647 3628 50.6 45 1.2 593 76.3 42.7 37 53.7 886 1906 18581 18599 51.4 47.4 4406 18697 18679 51.9 52.6 0.4 117 71 41 37 50.2 887 1907 18616 18636 51.4 47.6 4407 19216 19195 50.2 40.9 1.1 601 75.6 41.1 37 53.1 888 1908 3219 3238 50.7 50 4408 3503 3484 51.5 50 0.8 285 74.8 42.1 37 52.7 889 1909 18696 18715 51.7 50 4409 19216 19195 50.2 40.9 1.5 521 75.5 41.3 37 53 890 1910 3219 3238 50.7 50 4410 3504 3485 50.4 45 0.3 286 74.7 42 37 52.5 891 1911 27366 27384 52.2 52.6 4411 27573 27552 52.3 40.9 0.1 208 74.6 43.8 37 53 892 1912 27366 27384 52.2 52.6 4412 27567 27547 51.1 42.9 1 202 74.6 44.1 37 52.7 893 1913 3055 3075 51.8 47.6 4413 3646 3625 52 40.9 0.2 592 76.2 42.6 37 54 894 1914 18704 18724 50.8 47.6 4414 19216 19195 50.2 40.9 0.5 513 75.5 41.1 37 53 895 1915 16874 16893 52.1 50 4415 17056 17035 51.8 45.5 0.4 183 74.4 44.3 37 52.7 896 1916 12234 12252 50.6 47.4 4416 12739 12719 50.3 42.9 0.3 506 75.8 42.1 37 53.3 897 1917 7728 7746 51.7 52.6 4417 8054 8035 50.4 50 1.2 327 75 41.9 37 52.7 898 1918 15506 15527 50.8 40.9 4418 15647 15628 51 45 0.3 142 71.9 40.8 37 50.7 899 1919 985 1004 51.1 50 4419 1773 1755 51.7 52.6 0.6 789 76.7 43.1 37 54.1 900 1920 3217 3236 51.1 50 4420 3503 3484 51.5 50 0.4 287 74.8 42.2 37 52.8 901 1921 3791 3808 50 50 4421 4434 4416 51.5 52.6 1.4 644 75.4 40.4 37 52.9 902 1922 19794 19813 50 50 4422 19923 19904 50.1 50 0.1 130 72.7 43.8 37 51 903 1923 13039 13058 51.8 50 4423 13178 13157 50.4 40.9 1.5 140 73.8 45.7 37 51.9 904 1924 13033 13051 52.1 52.6 4424 13155 13138 50.4 50 1.7 123 73.7 47.2 37 51.8 905 1925 12233 12251 51.1 52.6 4425 12739 12719 50.3 42.9 0.9 507 75.9 42.2 37 53.3 906 1926 19795 19814 50.4 45 4426 19923 19903 50.9 47.6 0.4 129 72.5 43.4 37 51 907 1927 13177 13197 50.3 42.9 4427 13946 13929 51.5 50 1.2 770 75.9 41 37 53.3 908 1928 3799 3820 52.9 45.5 4428 4318 4294 54.4 40 1.5 520 75.4 41 37 53.7 909 1929 8867 8887 52.3 47.6 4429 9364 9346 53.9 52.6 1.6 498 75.8 42.2 37 53.9 910 1930 1472 1491 51.2 45 4430 2152 2133 50.7 45 0.5 681 76.5 42.9 37 53.8 911 1931 12233 12251 51.1 52.6 4431 12739 12718 51 40.9 0.2 507 75.9 42.2 37 53.5 912 1932 3055 3076 52.4 45.5 4432 3650 3631 53.1 50 0.7 596 76.3 42.8 37 54.2 913 1933 12726 12746 51.3 47.6 4433 13325 13305 50.5 47.6 0.7 600 76.7 43.7 37 53.9 914 1934 8867 8887 52.3 47.6 4434 9316 9296 50.8 42.9 1.5 450 75.4 41.6 37 53.1 915 1935 8867 8887 52.3 47.6 4435 9314 9295 51.1 50 1.2 448 75.5 41.7 37 53.3 916 1936 8867 8887 52.3 47.6 4436 9313 9294 50.4 50 1.9 447 75.5 41.6 37 53 917 1937 3055 3076 52.4 45.5 4437 3647 3628 50.6 45 1.8 593 76.3 42.7 37 53.7 918 1938 13176 13196 51.4 47.6 4438 13312 13294 51 52.6 0.4 137 72.9 43.8 37 51.5 919 1939 12726 12746 51.3 47.6 4439 13155 13138 50.4 50 0.9 430 76.4 44 37 53.7 920 1940 13701 13724 53.1 41.7 4440 14058 14040 51.4 52.6 1.7 358 74.5 40.2 37 52.7 921 1941 8372 8390 50.7 47.4 4441 9101 9081 50.5 47.6 0.2 730 75.5 40.3 37 53.1 922 1942 3055 3076 52.4 45.5 4442 3646 3625 52 40.9 0.4 592 76.2 42.6 37 54.1 923 1943 887 905 50.1 47.4 4443 1493 1474 50.8 45 0.7 607 77.1 44.6 37 54.1 924 1944 1046 1063 50.3 50 4444 1697 1677 51 42.9 0.7 652 76.9 43.9 37 54 925 1945 27378 27397 50.5 45 4445 27675 27656 50 40 0.5 298 74.1 40.3 37 52 926 1946 27378 27397 50.5 45 4446 27674 27654 51.9 42.9 1.4 297 74.2 40.4 37 52.2 927 1947 2671 2692 52.1 40.9 4447 3056 3037 52.1 55 0 386 74.8 40.7 37 53.1 928 1948 1046 1063 50.3 50 4448 1697 1678 50.3 45 0.1 652 76.9 43.9 37 54 929 1949 2387 2405 51.6 52.6 4449 2672 2654 50.9 52.6 0.8 286 77 47.6 37 54.3 930 1950 3792 3810 52.9 52.6 4450 4565 4542 53.9 41.7 1 774 75.6 40.3 37 53.8 931 1951 15506 15527 50.8 40.9 4451 15647 15629 50.3 47.4 0.5 142 71.9 40.8 37 50.5 932 1952 8794 8813 51.6 45 4452 9560 9540 51.6 42.9 0 767 75.9 41.2 37 53.7 933 1953 19801 19819 53.2 52.6 4453 19909 19885 52.5 40 0.7 109 71.4 43.1 37 50.8 934 1954 19988 20006 50.4 47.4 4454 20615 20597 50.6 47.4 0.2 628 75.3 40.1 37 52.9 935 1955 19991 20009 52.8 52.6 4455 20616 20597 52.3 45 0.5 626 75.3 40.1 37 53.5 936 1956 16875 16895 51.6 47.6 4456 17060 17041 51.1 50 0.5 186 74.6 44.6 37 52.6 937 1957 16875 16895 51.6 47.6 4457 17059 17039 50.6 47.6 1 185 74.4 44.3 37 52.4 938 1958 16875 16895 51.6 47.6 4458 17056 17035 51.8 45.5 0.2 182 74.2 44 37 52.5 939 1959 27442 27461 51.5 40 4459 27541 27521 51.7 47.6 0.1 100 71.2 44 37 50.4 940 1960 16875 16896 52.2 45.5 4460 17060 17041 51.1 50 1.1 186 74.6 44.6 37 52.6 941 1961 23841 23859 50.5 52.6 4461 24527 24507 51 42.9 0.5 687 76.1 41.9 37 53.5 942 1962 23841 23859 50.5 52.6 4462 24093 24075 50.9 52.6 0.4 253 76 45.8 37 53.5 943 1963 16875 16896 52.2 45.5 4463 17059 17039 50.6 47.6 1.6 185 74.4 44.3 37 52.4 944 1964 16875 16896 52.2 45.5 4464 17056 17035 51.8 45.5 0.5 182 74.2 44 37 52.6 945 1965 23843 23863 50.3 42.9 4465 24093 24075 50.9 52.6 0.5 251 75.8 45.4 37 53.3 946 1966 16875 16896 52.2 45.5 4466 17041 17023 53.5 52.6 1.3 167 73.8 43.7 37 52.4 947 1967 28187 28205 53.1 52.6 4467 28673 28654 53.5 55 0.5 487 80 52.4 37 57 948 1968 28190 28208 51.7 52.6 4468 28672 28654 50.6 52.6 1.2 483 79.9 52.2 37 56.2 949 1969 24030 24047 50.7 50 4469 24527 24508 50.5 45 0.2 498 75.4 41.2 37 53 950 1970 24031 24050 56.5 55 4470 24816 24792 54.7 40 1.8 786 76.3 42 37 54.9 951 1971 7880 7900 50.3 42.9 4471 8049 8032 50.4 50 0 170 72.8 41.2 37 51.2 952 1972 24096 24119 54.4 41.7 4472 24815 24791 54.5 40 0.1 720 75.8 41 37 54.5 953 1973 17790 17811 51.6 40.9 4473 18233 18214 52 50 0.4 444 75.1 40.8 37 53.2 954 1974 24174 24194 50.9 42.9 4474 24938 24921 50.4 50 0.5 765 75.8 40.9 37 53.3 955 1975 16875 16896 52.2 45.5 4475 17039 17022 51.4 50 0.8 165 73.7 43.6 37 52.1 956 1976 24174 24195 52.5 40.9 4476 24936 24919 51.8 50 0.7 763 75.8 41 37 53.7 957 1977 24179 24198 51 45 4477 24938 24921 50.4 50 0.6 760 75.8 40.9 37 53.3 958 1978 16875 16896 52.2 45.5 4478 17038 17021 50.7 50 1.6 164 73.8 43.9 37 52 959 1979 24180 24199 50.3 40 4479 24936 24919 51.8 50 1.5 757 75.8 41 37 53.2 960 1980 2823 2844 50.4 45.5 4480 3186 3165 50.4 40.9 0 364 75.5 42.6 37 53.1 961 1981 10142 10163 51.3 40.9 4481 10608 10589 51 50 0.3 467 74.9 40 37 52.8 962 1982 1046 1063 50.3 50 4482 1483 1464 51.3 45 0.9 438 76.2 43.6 37 53.6 963 1983 17388 17408 50.7 42.9 4483 17501 17481 51.2 42.9 0.6 114 70.5 40.4 37 49.7 964 1984 24179 24200 53.3 40.9 4484 24740 24717 52.5 41.7 0.8 562 76 42.2 37 54 965 1985 24379 24398 55 55 4485 25088 25070 54.5 52.6 0.5 710 75.9 41.3 37 54.6 966 1986 24379 24398 55 55 4486 25087 25069 53.7 52.6 1.3 709 75.9 41.3 37 54.3 967 1987 16874 16893 52.1 50 4487 17059 17039 50.6 47.6 1.5 186 74.6 44.6 37 52.5 968 1988 24380 24399 55 55 4488 25088 25070 54.5 52.6 0.5 709 75.9 41.3 37 54.6 969 1989 28522 28542 50.2 42.9 4489 28671 28653 50.2 52.6 0 150 76.2 50.7 37 53.5 970 1990 16874 16893 52.1 50 4490 17060 17041 51.1 50 1 187 74.7 44.9 37 52.7 971 1991 24380 24399 55 55 4491 25087 25069 53.7 52.6 1.3 708 75.9 41.4 37 54.4 972 1992 17608 17627 50.2 45 4492 18239 18220 50 45 0.2 632 75.3 40.2 37 52.8 973 1993 17608 17627 50.2 45 4493 18238 18219 50.3 45 0.1 631 75.3 40.3 37 52.9 974 1994 1046 1063 50.3 50 4494 1483 1465 50.5 47.4 0.2 438 76.2 43.6 37 53.6 975 1995 17608 17628 50.9 42.9 4495 18239 18220 50 45 0.9 632 75.3 40.2 37 52.8 976 1996 17608 17628 50.9 42.9 4496 18238 18219 50.3 45 0.7 631 75.3 40.3 37 52.9 977 1997 8063 8084 51.4 45.5 4497 8188 8169 50.5 45 0.9 126 72.1 42.9 37 50.7 978 1998 12236 12256 51.2 42.9 4498 12739 12718 51 40.9 0.2 504 75.8 42.1 37 53.5 979 1999 13176 13196 51.4 47.6 4499 13325 13305 50.5 47.6 0.8 150 73.4 44 37 51.7 980 2000 2371 2389 50.3 47.4 4500 2749 2728 50.3 45.5 0 379 76.9 45.9 37 54.1 981 2001 9402 9420 51.3 47.4 4501 9989 9968 51 40.9 0.4 588 75.4 40.5 37 53.1 982 2002 9931 9950 50.2 45 4502 10183 10166 50.9 50 0.7 253 75.2 43.9 37 52.8 983 2003 2387 2405 51.6 52.6 4503 2997 2976 51.4 40.9 0.2 611 76.6 43.5 37 54.2 984 2004 3788 3805 50 50 4504 4435 4417 50.5 52.6 0.4 648 75.4 40.4 37 52.9 985 2005 26039 26057 52.6 52.6 4505 26650 26630 51.4 42.9 1.2 612 75.3 40.4 37 53.3 986 2006 2371 2389 50.3 47.4 4506 3053 3034 50.3 50 0 683 76.7 43.3 37 53.9 987 2007 3 21 53.4 52.6 4507 315 296 51.9 50 1.5 313 76.9 46.6 37 54.5 988 2008 2371 2389 50.3 47.4 4508 3056 3037 52.1 55 1.7 686 76.7 43.4 37 53.9 989 2009 13040 13059 50.9 50 4509 13155 13137 52.1 52.6 1.2 116 73.2 46.6 37 51.6 990 2010 9931 9950 50.2 45 4510 10183 10165 51.7 47.4 1.5 253 75.2 43.9 37 52.8 991 2011 3788 3805 50 50 4511 4434 4416 51.5 52.6 1.4 647 75.4 40.3 37 52.9 992 2012 13176 13196 51.4 47.6 4512 13946 13929 51.5 50 0.2 771 75.9 41.1 37 53.6 993 2013 3772 3792 51.2 42.9 4513 4444 4424 50.6 42.9 0.7 673 75.6 40.7 37 53.2 994 2014 13176 13196 51.4 47.6 4514 13320 13300 51.4 47.6 0 145 73.3 44.1 37 51.9 995 2015 8861 8880 50.2 45 4515 9245 9226 50 45 0.1 385 74.9 40.8 37 52.5 996 2016 8868 8889 50.4 40.9 4516 9310 9291 51.2 45 0.8 443 75.3 41.3 37 52.9 997 2017 16366 16384 50.3 52.6 4517 16774 16752 52.2 43.5 1.9 409 75.1 41.1 37 52.8 998 2018 9934 9953 50.7 50 4518 10183 10166 50.9 50 0.2 250 75.2 44 37 53 999 2019 9055 9079 52.8 40 4519 9342 9323 52.1 50 0.8 288 75.1 42.7 37 53.3 1000 2020 8868 8889 50.4 40.9 4520 9249 9231 50.8 47.4 0.4 382 75.1 41.4 37 52.8 1001 2021 16366 16385 52.9 55 4521 16774 16752 52.2 43.5 0.6 409 75.1 41.1 37 53.3 1002 2022 8868 8889 50.4 40.9 4522 9249 9230 51.5 45 1.1 382 75.1 41.4 37 52.8 1003 2023 9934 9953 50.7 50 4523 10183 10165 51.7 47.4 0.9 250 75.2 44 37 53 1004 2024 25772 25793 52.4 40.9 4524 26183 26163 51.7 42.9 0.7 412 74.8 40.3 37 53 1005 2025 13039 13057 51.1 52.6 4525 13155 13138 50.4 50 0.7 117 73.4 47 37 51.6 1006 2026 25771 25790 51.1 45 4526 26183 26164 51 45 0.1 413 74.8 40.4 37 52.8 1007 2027 25769 25786 50.3 50 4527 26183 26163 51.7 42.9 1.4 415 74.9 40.5 37 52.6 1008 2028 3794 3812 52.9 52.6 4528 4436 4417 52.2 50 0.6 643 75.4 40.4 37 53.6 1009 2029 887 905 50.1 47.4 4529 1480 1462 51.6 47.4 1.5 594 77.1 44.6 37 54.1 1010 2030 3794 3812 52.9 52.6 4530 4434 4416 51.5 52.6 1.4 641 75.4 40.4 37 53.3 1011 2031 12370 12388 50.1 47.4 4531 12994 12976 50.3 47.4 0.3 625 76.4 42.9 37 53.6 1012 2032 3797 3815 50.9 47.4 4532 4186 4168 51.8 52.6 0.9 390 75.3 41.8 37 53.1 1013 2033 3795 3813 52.1 52.6 4533 4435 4417 50.5 52.6 1.6 641 75.5 40.6 37 53.1 1014 2034 3795 3813 52.1 52.6 4534 4434 4416 51.5 52.6 0.6 640 75.4 40.5 37 53.3 1015 2035 13177 13197 50.3 42.9 4535 13323 13304 51.1 45 0.8 147 73.2 43.5 37 51.4 1016 2036 1046 1064 51.2 47.4 4536 1401 1382 50.6 45 0.6 356 75.6 43 37 53.2 1017 2037 16549 16567 54.9 52.6 4537 17057 17035 53 43.5 1.9 509 75.9 42.2 37 54.1 1018 2038 1046 1064 51.2 47.4 4538 1483 1464 51.3 45 0.1 438 76.2 43.6 37 53.8 1019 2039 16551 16568 51.1 50 4539 17056 17035 51.8 45.5 0.7 506 75.9 42.3 37 53.6 1020 2040 1046 1064 51.2 47.4 4540 1483 1465 50.5 47.4 0.6 438 76.2 43.6 37 53.6 1021 2041 1046 1064 51.2 47.4 4541 1484 1466 53.1 52.6 2 439 76.3 43.7 37 53.9 1022 2042 1046 1064 51.2 47.4 4542 1697 1676 51.7 40.9 0.5 652 76.9 43.9 37 54.2 1023 2043 1046 1064 51.2 47.4 4543 1697 1677 51 42.9 0.2 652 76.9 43.9 37 54.2 1024 2044 16555 16572 50.3 50 4544 17111 17090 51.1 40.9 0.8 557 76.1 42.5 37 53.5 1025 2045 1046 1064 51.2 47.4 4545 1697 1678 50.3 45 0.9 652 76.9 43.9 37 54 1026 2046 1046 1063 50.3 50 4546 1401 1382 50.6 45 0.2 356 75.6 43 37 53.1 1027 2047 3796 3814 50.8 52.6 4547 4435 4417 50.5 52.6 0.3 640 75.4 40.5 37 53.1 1028 2048 12232 12250 51.9 52.6 4548 12993 12975 51.4 47.4 0.5 762 76.5 42.5 37 54 1029 2049 12236 12256 51.2 42.9 4549 12739 12719 50.3 42.9 0.9 504 75.8 42.1 37 53.2 1030 2050 28937 28956 52.4 50 4550 29306 29288 53.5 52.6 1.1 370 76.6 45.1 37 54.4 1031 2051 12232 12250 51.9 52.6 4551 12996 12977 50.2 40 1.7 765 76.4 42.4 37 53.6 1032 2052 3234 3254 51.1 47.6 4552 3504 3485 50.4 45 0.7 271 74.4 41.3 37 52.3 1033 2053 3234 3254 51.1 47.6 4553 3503 3484 51.5 50 0.4 270 74.4 41.5 37 52.5 1034 2054 9922 9941 51.3 50 4554 10670 10649 51.3 40.9 0.1 749 75.8 40.9 37 53.5 1035 2055 3792 3810 52.9 52.6 4555 4434 4416 51.5 52.6 1.4 643 75.4 40.4 37 53.3 1036 2056 3234 3254 51.1 47.6 4556 3494 3473 50.4 40.9 0.6 261 74.1 41 37 52.1 1037 2057 4255 4276 51.7 45.5 4557 4608 4590 51.5 52.6 0.2 354 74.7 40.7 37 52.8 1038 2058 24562 24580 50.1 52.6 4558 24936 24919 51.8 50 1.7 375 75.6 42.7 37 53 1039 2059 24562 24580 50.1 52.6 4559 24938 24921 50.4 50 0.3 377 75.5 42.4 37 53 1040 2060 24562 24580 50.1 52.6 4560 25182 25164 51.4 47.4 1.3 621 75.9 41.7 37 53.3 1041 2061 24559 24579 52 52.4 4561 24936 24919 51.8 50 0.2 378 75.7 42.9 37 53.6 1042 2062 24559 24579 52 52.4 4562 24938 24921 50.4 50 1.6 380 75.6 42.6 37 53.1 1043 2063 1046 1063 50.3 50 4563 1697 1676 51.7 40.9 1.3 652 76.9 43.9 37 54 1044 2064 24482 24503 51.6 40.9 4564 24815 24792 53.4 41.7 1.8 334 75.4 42.8 37 53.4 1045 2065 13177 13197 50.3 42.9 4565 13326 13306 50.7 42.9 0.4 150 73.2 43.3 37 51.4 1046 2066 24480 24502 54.2 47.8 4566 24815 24791 54.5 40 0.3 336 75.6 43.2 37 54.3 1047 2067 17840 17859 50.8 45 4567 18223 18206 51.8 50 1 384 74.7 40.4 37 52.6 1048 2068 24480 24500 53.2 47.6 4568 24815 24792 53.4 41.7 0.2 336 75.6 43.2 37 54 1049 2069 17840 17859 50.8 45 4569 18231 18210 52.2 45.5 1.4 392 74.8 40.6 37 52.7 1050 2070 28821 28840 51.8 45 4570 29298 29279 52.6 55 0.8 478 77 45.2 37 54.6 1051 2071 24418 24440 55 47.8 4571 24517 24494 53.2 41.7 1.8 100 70.8 43 37 50.6 1052 2072 13701 13722 50.4 40.9 4572 14058 14040 51.4 52.6 1 358 74.5 40.2 37 52.4 1053 2073 28821 28840 51.8 45 4573 29358 29339 52.8 50 1 538 77.1 45 37 54.6 1054 2074 17792 17813 51.6 40.9 4574 18233 18214 52 50 0.4 442 75.1 40.7 37 53.1 1055 2075 24420 24440 50.8 42.9 4575 25079 25061 52.7 52.6 1.9 660 75.7 41.1 37 53.3 1056 2076 28821 28839 51.1 47.4 4576 29298 29279 52.6 55 1.5 478 77 45.2 37 54.3 1057 2077 3796 3814 50.8 52.6 4577 4434 4416 51.5 52.6 0.7 639 75.4 40.4 37 53.1 1058 2078 28821 28839 51.1 47.4 4578 29358 29339 52.8 50 1.7 538 77.1 45 37 54.4 1059 2079 28820 28838 53.7 52.6 4579 29298 29279 52.6 55 1.1 479 77.1 45.3 37 54.8 1060 2080 24418 24439 52.9 45.5 4580 25079 25061 52.7 52.6 0.2 662 75.8 41.2 37 54 1061 2081 28820 28838 53.7 52.6 4581 29358 29339 52.8 50 0.9 539 77.1 45.1 37 54.9 1062 2082 27369 27389 52.5 47.6 4582 27468 27451 51.1 50 1.4 100 71.2 44 38 50.3 1063 2083 7725 7742 50 50 4583 8187 8167 50.4 42.9 0.3 463 75.6 41.9 38 53 1064 2084 16549 16567 54.9 52.6 4584 17040 17021 53.4 50 1.5 492 75.9 42.3 38 54.2 1065 2085 3221 3239 51.5 52.6 4585 3500 3481 51.2 50 0.3 280 74.6 41.8 38 52.7 1066 2086 16549 16567 54.9 52.6 4586 17041 17022 54.1 50 0.8 493 75.8 42.2 38 54.4 1067 2087 20138 20158 50.1 42.9 4587 20615 20597 50.6 47.4 0.5 478 75 40.4 38 52.7 1068 2088 20078 20099 50.5 40.9 4588 20615 20597 50.6 47.4 0.1 538 75.3 40.5 38 52.9 1069 2089 13039 13057 51.1 52.6 4589 13325 13305 50.5 47.6 0.6 287 75.8 44.6 38 53.3 1070 2090 16549 16567 54.9 52.6 4590 17041 17023 53.5 52.6 1.4 493 75.8 42.2 38 54.2 1071 2091 13701 13725 53.6 40 4591 14124 14106 52.4 52.6 1.2 424 75.1 41 38 53.4 1072 2092 12975 12993 51.4 47.4 4592 13320 13300 51.4 47.6 0 346 76.1 44.2 38 53.8 1073 2093 16548 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530 75.3 40.6 38 52.9 1086 2106 3360 3380 51.4 42.9 4606 3497 3478 51.3 50 0.1 138 74 46.4 38 52.3 1087 2107 9926 9944 50.5 52.6 4607 10455 10435 50.5 42.9 0 530 75.3 40.6 38 52.9 1088 2108 10140 10159 52.4 50 4608 10608 10589 51 50 1.4 469 75 40.3 38 52.9 1089 2109 9931 9950 50.2 45 4609 10455 10435 50.5 42.9 0.3 525 75.3 40.6 38 52.8 1090 2110 3219 3238 50.7 50 4610 3500 3481 51.2 50 0.5 282 74.7 41.8 38 52.6 1091 2111 3219 3238 50.7 50 4611 3497 3478 51.3 50 0.6 279 74.7 41.9 38 52.6 1092 2112 3360 3380 51.4 42.9 4612 3500 3481 51.2 50 0.3 141 74 46.1 38 52.3 1093 2113 3360 3380 51.4 42.9 4613 3503 3484 51.5 50 0 144 74.3 46.5 38 52.5 1094 2114 2223 2244 51.4 45.5 4614 2672 2653 51.6 50 0.2 450 77 45.3 38 54.4 1095 2115 9922 9941 51.3 50 4615 10449 10428 51.9 40.9 0.7 528 75.4 40.9 38 53.3 1096 2116 13039 13057 51.1 52.6 4616 13312 13294 51 52.6 0.1 274 75.7 44.5 38 53.4 1097 2117 15951 15973 52.1 43.5 4617 16174 16154 50.4 42.9 1.7 224 73.5 40.6 38 51.7 1098 2118 13176 13196 51.4 47.6 4618 13545 13526 52.9 55 1.5 370 77 46.2 38 54.4 1099 2119 11541 11562 51.5 40.9 4619 11983 11965 53 52.6 1.5 443 75 40.6 38 53.1 1100 2120 2429 2447 50.2 47.4 4620 3056 3038 50.8 52.6 0.6 628 76.3 42.7 38 53.6 1101 2121 11545 11563 50.8 47.4 4621 12258 12238 50.3 42.9 0.5 714 76.2 42 38 53.5 1102 2122 8868 8889 50.4 40.9 4622 9245 9226 50 45 0.4 378 74.9 41 38 52.6 1103 2123 27361 27380 52.4 55 4623 27466 27448 52.3 52.6 0.1 106 72.5 46.2 38 51.5 1104 2124 8861 8880 50.2 45 4624 9340 9319 50.8 45.5 0.6 480 75.5 41.5 38 53 1105 2125 1784 1802 51.8 52.6 4625 2113 2094 50.1 45 1.7 330 76 44.2 38 53.3 1106 2126 8868 8889 50.4 40.9 4626 9107 9086 51.6 45.5 1.2 240 74 41.2 38 52 1107 2127 19795 19814 50.4 45 4627 20099 20078 50.5 40.9 0 305 74.4 40.7 38 52.3 1108 2128 26708 26731 54.2 41.7 4628 27347 27324 52.3 41.7 1.9 640 75.6 40.8 38 53.7 1109 2129 19794 19813 50 50 4629 19920 19899 50.2 40.9 0.2 127 72.3 43.3 38 50.7 1110 2130 3031 3051 51.3 52.4 4630 3650 3631 53.1 50 1.8 620 76.5 43.1 38 54 1111 2131 3031 3051 51.3 52.4 4631 3647 3628 50.6 45 0.7 617 76.4 42.9 38 53.8 1112 2132 19794 19813 50 50 4632 19922 19902 50 42.9 0 129 72.5 43.4 38 50.8 1113 2133 12236 12256 51.2 42.9 4633 12994 12976 50.3 47.4 0.8 759 76.4 42.4 38 53.7 1114 2134 26708 26731 54.2 41.7 4634 27467 27449 52.8 47.4 1.4 760 76 41.3 38 54.1 1115 2135 19716 19737 52.2 45.5 4635 19922 19901 51.5 45.5 0.7 207 73.5 41.1 38 52 1116 2136 19715 19735 52.5 47.6 4636 19922 19901 51.5 45.5 0.9 208 73.6 41.3 38 52.1 1117 2137 3360 3379 50.7 45 4637 3503 3484 51.5 50 0.7 144 74.3 46.5 38 52.3 1118 2138 9055 19079 52.8 40 4638 9364 9346 53.9 52.6 1.1 310 75.3 42.9 38 53.7 1119 2139 1782 1801 52.7 50 4639 1881 1861 54.5 52.4 1.8 100 72.4 47 38 51.6 1120 2140 26708 26727 50 45 4640 27468 27450 51.9 47.4 1.8 761 75.9 41.3 38 53.3 1121 2141 26708 26727 50 45 4641 27468 27451 51.1 50 1.1 761 75.9 41.3 38 53.3 1122 2142 4593 4613 51.5 47.6 4642 4995 4975 51.7 42.9 0.1 403 76 43.4 38 53.8 1123 2143 19709 19730 51.3 40.9 4643 19930 19911 50.7 50 0.5 222 74 41.9 38 52.1 1124 2144 26421 26441 51.5 42.9 4644 26587 26570 50.2 50 1.3 167 72.3 40.1 38 50.8 1125 2145 18979 19000 51.6 45.5 4645 19217 19195 51.7 43.5 0 239 73.5 40.2 38 52.1 1126 2146 18703 18724 53.5 50 4646 19476 19453 53.5 41.7 0 774 75.6 40.3 38 54 1127 2147 4255 4276 51.7 45.5 4647 4708 4690 50.3 47.4 1.4 454 75.1 40.7 38 52.8 1128 2148 3232 3252 51.1 47.6 4648 3503 3484 51.5 50 0.4 272 74.6 41.9 38 52.6 1129 2149 26421 26441 51.5 42.9 4649 26656 26636 51.3 47.6 0.2 236 74.2 41.9 38 52.5 1130 2150 3232 3252 51.1 47.6 4650 3504 3485 50.4 45 0.7 273 74.6 41.8 38 52.4 1131 2151 26421 26441 51.5 42.9 4651 26660 26641 50.2 50 1.3 240 74.2 41.7 38 52.1 1132 2152 26421 26441 51.5 42.9 4652 26683 26665 52.7 52.6 1.2 263 74.8 42.6 38 52.9 1133 2153 26421 26441 51.5 42.9 4653 26686 26669 50.5 50 0.9 266 74.8 42.5 38 52.6 1134 2154 26421 26441 51.5 42.9 4654 26691 26673 51.3 47.4 0.1 271 74.8 42.4 38 52.9 1135 2155 18704 18724 50.8 47.6 4655 19476 19456 50.5 42.9 0.3 773 75.5 40.2 38 53.1 1136 2156 18704 18724 50.8 47.6 4656 19482 19463 50.1 45 0.7 779 75.5 40.2 38 53 1137 2157 942 960 52.1 52.6 4657 1498 1481 51 50 1.1 557 76.9 44.5 38 54.3 1138 2158 942 960 52.1 52.6 4658 1497 1480 50.3 50 1.9 556 77 44.6 38 54.1 1139 2159 13040 13059 50.9 50 4659 13312 13294 51 52.6 0.1 273 75.6 44.3 38 53.3 1140 2160 18696 18715 51.7 50 4660 19476 19453 53.5 41.7 1.8 781 75.6 40.3 38 53.5 1141 2161 18696 18715 51.7 50 4661 19476 19456 50.5 42.9 1.3 781 75.6 40.3 38 53.1 1142 2162 3232 3251 50.3 50 4662 3503 3484 51.5 50 1.1 272 74.6 41.9 38 52.4 1143 2163 3031 3051 51.3 52.4 4663 3646 3625 52 40.9 0.7 616 76.4 42.9 38 54 1144 2164 9130 9150 51.3 42.9 4664 9560 9541 50.9 45 0.4 431 75.3 41.3 38 53 1145 2165 18224 18243 53.1 50 4665 18696 18672 53.9 40 0.8 473 75.7 42.1 38 54 1146 2166 18224 18243 53.1 50 4666 18696 18673 53.4 41.7 0.3 473 75.7 42.1 38 54 1147 2167 18225 18243 51.4 52.6 4667 18697 18679 51.9 52.6 0.5 473 75.8 42.3 38 53.6 1148 2168 9130 9150 51.3 42.9 4668 9560 9540 51.6 42.9 0.3 431 75.3 41.3 38 53.2 1149 2169 8866 8885 51.1 45 4669 9252 9235 50.1 50 1 387 75.1 41.3 38 52.7 1150 2170 9130 9150 51.3 42.9 4670 9559 9539 50.6 42.9 0.7 430 75.3 41.4 38 53 1151 2171 12267 12290 54.5 41.7 4671 12501 12480 53.5 45.5 1 235 74.3 42.1 38 53.2 1152 2172 3427 3446 52.7 50 4672 3650 3631 53.1 50 0.4 224 74.3 42.4 38 52.9 1153 2173 3427 3446 52.7 50 4673 3648 3628 52.3 42.9 0.4 222 74 41.9 38 52.6 1154 2174 26039 26058 54 55 4674 26184 26164 52.4 42.9 1.6 146 71.8 40.4 38 51.1 1155 2175 3230 3249 50.1 45 4675 3503 3484 51.5 50 1.4 274 74.5 41.6 38 52.3 1156 2176 3230 3249 50.1 45 4676 3504 3485 50.4 45 0.3 275 74.4 41.5 38 52.2 1157 2177 3427 3446 52.7 50 4677 3646 3625 52 40.9 0.6 220 74 41.8 38 52.5 1158 2178 3429 3449 50.4 42.9 4678 3647 3628 50.6 45 0.2 219 73.9 41.6 38 51.9 1159 2179 3429 3449 50.4 42.9 4679 3646 3625 52 40.9 1.6 218 73.7 41.3 38 51.8 1160 2180 8866 8885 51.1 45 4680 9249 9231 50.8 47.4 0.3 384 75.1 41.4 38 52.9 1161 2181 3428 3449 52.8 45.5 4681 3650 3631 53.1 50 0.3 223 74.2 42.2 38 52.8 1162 2182 18077 18098 52.9 50 4682 18696 18672 53.9 40 1 620 76.2 42.4 38 54.3 1163 2183 18078 18098 51.5 47.6 4683 18696 18673 53.4 41.7 1.9 619 76.2 42.3 38 53.9 1164 2184 8866 8885 51.1 45 4684 9249 9230 51.5 45 0.4 384 75.1 41.4 38 53 1165 2185 3229 3248 50.6 50 4685 3503 3484 51.5 50 0.8 275 74.6 41.8 38 52.5 1166 2186 12267 12290 54.5 41.7 4686 12495 12476 52.7 45 1.9 229 74.1 41.9 38 52.8 1167 2187 8220 8240 54 47.6 4687 8929 8910 54.5 55 0.4 710 75.4 40 38 54.1 1168 2188 18080 18098 51.2 52.6 4688 18238 18219 50.3 45 0.9 159 74 44.7 38 52 1169 2189 18080 18098 51.2 52.6 4689 18239 18220 50 45 1.2 160 73.9 44.4 38 51.8 1170 2190 18080 18098 51.2 52.6 4690 18697 18679 51.9 52.6 0.7 618 76.3 42.6 38 53.8 1171 2191 18076 18097 53.1 45.5 4691 18712 18693 54.8 55 1.7 637 76.3 42.5 38 54.4 1172 2192 8866 8885 51.1 45 4692 9245 9226 50 45 1.1 380 75 41.1 38 52.6 1173 2193 943 961 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38 52.3 1236 2256 29182 29206 55.4 44 4756 29301 29282 55.3 55 0.1 120 73.4 46.7 38 53.1 1237 2257 29183 29206 52.9 41.7 4757 29306 29287 54.6 55 1.7 124 73.3 46 38 52.3 1238 2258 29186 29206 51.3 42.9 4758 29298 29279 52.6 55 1.3 113 72.8 46 38 51.5 1239 2259 16979 17000 52.6 50 4759 17483 17465 54.4 52.6 1.8 505 75.9 42.2 38 54 1240 2260 29182 29205 54.6 41.7 4760 29298 29279 52.6 55 1.9 117 73.1 46.2 38 52 1241 2261 16981 17000 51.3 50 4761 17111 17090 51.1 40.9 0.2 131 74.5 48.1 38 52.6 1242 2262 13177 13197 50.3 42.9 4762 13949 13932 51.6 50 1.3 773 75.8 41 38 53.3 1243 2263 8867 8887 52.3 47.6 4763 9252 9234 51.4 52.6 0.9 386 75.1 41.5 38 53.1 1244 2264 24420 24440 50.8 42.9 4764 25081 25063 52.4 52.6 1.6 662 75.7 40.9 38 53.3 1245 2265 7727 7745 50.8 47.4 4765 8188 8169 50.5 45 0.4 462 75.6 41.8 38 53.1 1246 2266 2387 2405 51.6 52.6 4766 3055 3036 50.6 50 1.1 669 76.7 43.3 38 53.9 1247 2267 2671 2692 52.1 40.9 4767 3055 3036 50.6 50 1.5 385 74.8 40.5 38 52.6 1248 2268 29182 29202 51.2 42.9 4768 29298 29279 52.6 55 1.4 117 73.1 46.2 38 51.6 1249 2269 24418 24439 52.9 45.5 4769 25081 25063 52.4 52.6 0.5 664 75.7 41.1 38 53.8 1250 2270 12373 12391 50.8 47.4 4770 12992 12974 51.2 52.6 0.4 620 76.5 43.1 38 53.9 1251 2271 29179 29199 51.4 42.9 4771 29298 29279 52.6 55 1.2 120 73.4 46.7 38 51.9 1252 2272 1783 1803 54.2 47.6 4772 1882 1861 56 50 1.8 100 72.4 47 38 52 1253 2273 12373 12391 50.8 47.4 4773 12498 12480 50 47.4 0.7 126 72.8 44.4 38 51 1254 2274 7728 7746 51.7 52.6 4774 8190 8172 50.3 47.4 1.4 463 75.6 41.9 38 53.1 1255 2275 16875 16896 52.2 45.5 4775 17064 17045 51.4 50 0.8 190 74.5 44.2 38 52.7 1256 2276 1402 1425 52.8 41.7 4776 2103 2082 52 45.5 0.8 702 76.7 43.3 38 54.4 1257 2277 28971 28993 51.9 43.5 4777 29358 29339 52.8 50 0.9 388 76.3 44.3 38 54.1 1258 2278 24380 24399 55 55 4778 25080 25061 54.1 50 1 701 75.9 41.4 38 54.5 1259 2279 24380 24399 55 55 4779 25080 25062 53.5 52.6 1.6 701 75.9 41.4 38 54.3 1260 2280 3168 3189 51 45.5 4780 3497 3478 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1347 2367 16378 16397 50.4 45 4867 16777 16758 51.5 50 1 400 75 41 39 52.7 1348 2368 16378 16397 50.4 45 4868 16775 16756 50.3 45 0.1 398 75 41 39 52.7 1349 2369 16378 16397 50.4 45 4869 16775 16755 51.1 42.9 0.6 398 75 41 39 52.7 1350 2370 16378 16397 50.4 45 4870 16774 16754 50.4 42.9 0 397 75 41.1 39 52.7 1351 2371 16378 16397 50.4 45 4871 16774 16753 51.1 40.9 0.7 397 75 41.1 39 52.8 1352 2372 16378 16397 50.4 45 4872 16774 16752 52.2 43.5 1.8 397 75 41.1 39 52.8 1353 2373 10250 10274 51.6 40 4873 10608 10589 51 50 0.6 359 74.6 40.4 39 52.6 1354 2374 16548 16566 54.9 52.6 4874 17112 17090 53.3 43.5 1.6 565 76.3 42.8 39 54.5 1355 2375 19709 19730 51.3 40.9 4875 19922 19902 50 42.9 1.2 214 73.8 41.6 39 51.8 1356 2376 3218 3237 50.5 45 4876 3504 3485 50.4 45 0.1 287 74.7 41.8 39 52.5 1357 2377 3218 3237 50.5 45 4877 3503 3484 51.5 50 0.9 286 74.7 42 39 52.6 1358 2378 19709 19730 51.3 40.9 4878 19920 19899 50.2 40.9 1.1 212 73.7 41.5 39 51.8 1359 2379 1402 1422 50.2 42.9 4879 1501 1480 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73.9 42.6 40 52.4 1732 2752 8376 8396 50.6 42.9 5252 9107 9086 51.6 45.5 1 732 75.4 40 40 53.1 1733 2753 11541 11561 50.9 42.9 5253 11727 11708 50.4 45 0.5 187 73 40.6 40 51.3 1734 2754 11540 11561 53.8 45.5 5254 11984 11966 53 52.6 0.7 445 75.1 40.7 40 53.6 1735 2755 2371 2389 50.3 47.4 5255 2672 2654 50.9 52.6 0.5 302 77.1 47.4 40 54.2 1736 2756 2371 2389 50.3 47.4 5256 2998 2977 51.1 40.9 0.8 628 76.7 43.5 40 53.9 1737 2757 11543 11562 50.4 40 5257 11727 11708 50.4 45 0.1 185 72.9 40.5 40 51.2 1738 2758 26040 26061 56.4 54.5 5258 26589 26567 56.1 47.8 0.4 550 75.1 40 40 54.5 1739 2759 11541 11562 51.5 40.9 5259 11984 11966 53 52.6 1.5 444 75 40.5 40 53.1 1740 2760 7728 7746 51.7 52.6 5260 8187 8167 50.4 42.9 1.3 460 75.6 42 40 53.2 1741 2761 26040 26061 56.4 54.5 5261 26657 26634 54.6 41.7 1.9 618 75.5 40.6 40 54.3 1742 2762 2223 2243 50.2 42.9 5262 2675 2656 50.4 50 0.2 453 77 45.3 40 54 1743 2763 2220 2239 51.3 45 5263 2676 2657 50.7 50 0.5 457 76.9 45.1 40 54.1 1744 2764 11541 11560 50.1 45 5264 11727 11707 51.1 42.9 1 187 73 40.6 40 51.2 1745 2765 24559 24580 54.2 54.5 5265 25088 25070 54.5 52.6 0.2 530 75.5 41.1 40 54.2 1746 2766 12233 12251 51.1 52.6 5266 12998 12979 50.1 45 1 766 76.5 42.6 40 53.7 1747 2767 12233 12251 51.1 52.6 5267 12412 12392 50 42.9 1.1 180 73.2 41.7 40 51.4 1748 2768 24562 24580 50.1 52.6 5268 25086 25069 50.3 50 0.2 525 75.4 41 40 52.9 1749 2769 9931 9950 50.2 45 5269 10608 10589 51 50 0.8 678 75.8 41.2 40 53.2 1750 2770 24562 24580 50.1 52.6 5270 25209 25188 52 45.5 1.9 648 76.1 42 40 53.4 1751 2771 24562 24580 50.1 52.6 5271 25209 25189 51.4 47.6 1.3 648 76.1 42 40 53.4 1752 2772 3789 3807 51.8 52.6 5272 4445 4425 50.6 42.9 1.2 657 75.5 40.5 40 53.1 1753 2773 26039 26058 54 55 5273 26656 26634 53.4 43.5 0.6 618 75.5 40.6 40 53.9 1754 2774 26039 26058 54 55 5274 26660 26639 52.5 45.5 1.5 622 75.4 40.5 40 53.7 1755 2775 3789 3807 51.8 52.6 5275 4444 4424 50.6 42.9 1.2 656 75.5 40.5 40 53.1 1756 2776 24559 24579 52 52.4 5276 25086 25069 50.3 50 1.6 528 75.5 41.1 40 53 1757 2777 12235 12253 50.1 52.6 5277 12999 12980 50.6 40 0.5 765 76.4 42.5 40 53.6 1758 2778 24559 24579 52 52.4 5278 25209 25188 52 45.5 0 651 76.1 42.1 40 54 1759 2779 24559 24579 52 52.4 5279 25209 25189 51.4 47.6 0.6 651 76.1 42.1 40 53.8 1760 2780 887 905 50.1 47.4 5280 1499 1482 50.1 50 0.1 613 77.1 44.7 40 54.1 1761 2781 24558 24577 50.7 50 5281 25182 25164 51.4 47.4 0.7 625 76 41.9 40 53.5 1762 2782 26039 26058 54 55 5282 26828 26810 52.9 52.6 1.2 790 76.4 42.4 40 54.5 1763 2783 8866 8885 51.1 45 5283 9597 9577 50.3 42.9 0.8 732 75.8 41.1 40 53.3 1764 2784 7727 7745 50.8 47.4 5284 8049 8032 50.4 50 0.5 323 74.8 41.5 40 52.6 1765 2785 13177 13197 50.3 42.9 5285 13747 13726 50.8 40.9 0.4 571 76.2 42.6 40 53.5 1766 2786 887 905 50.1 47.4 5286 1498 1481 51 50 0.9 612 77.2 44.8 40 54.1 1767 2787 1784 1803 52.5 50 5287 2103 2083 50.6 42.9 2 320 76 44.4 40 53.5 1768 2788 12235 12253 50.1 52.6 5288 12498 12480 50 47.4 0.1 264 74.7 42.4 40 52.4 1769 2789 1784 1802 51.8 52.6 5289 2103 2083 50.6 42.9 1.3 320 76 44.4 40 53.5 1770 2790 887 905 50.1 47.4 5290 1496 1478 50.4 47.4 0.3 610 77.1 44.8 40 54.1 1771 2791 1783 1801 52.9 52.6 5291 2153 2133 52.1 42.9 0.9 371 76 43.7 40 53.9 1772 2792 887 905 50.1 47.4 5292 1494 1476 50.7 47.4 0.6 608 77.1 44.7 40 54.1 1773 2793 3791 3809 51.8 52.6 5293 4445 4425 50.6 42.9 1.2 655 75.5 40.5 40 53.1 1774 2794 17813 17832 50.1 45 5294 18506 18488 51.2 52.6 1.2 694 75.8 41.2 40 53.2 1775 2795 3791 3809 51.8 52.6 5295 4444 4424 50.6 42.9 1.2 654 75.5 40.5 40 53.1 1776 2796 17840 17859 50.8 45 5296 18233 18215 51.3 52.6 0.5 394 74.9 40.9 40 52.8 1777 2797 17840 17859 50.8 45 5297 18233 18214 52 50 1.3 394 74.9 40.9 40 52.8 1778 2798 9930 9949 52.2 50 5298 10356 10336 52.4 47.6 0.2 427 75.6 42.2 40 53.7 1779 2799 15211 15230 50.2 45 5299 15949 15930 51.1 45 0.9 739 75.5 40.3 40 53 1780 2800 98 118 50.6 42.9 5300 253 233 51.8 47.6 1.2 156 74.5 46.2 40 52.4 1781 2801 18004 18023 51.1 50 5301 18233 18214 52 50 0.9 230 75 43.9 40 52.9 1782 2802 24420 24440 50.8 42.9 5302 24936 24919 51.8 50 1 517 75.9 42.2 40 53.5 1783 2803 24420 24440 50.8 42.9 5303 24938 24921 50.4 50 0.4 519 75.8 42 40 53.3 1784 2804 98 118 50.6 42.9 5304 254 235 50 45 0.6 157 74.4 45.9 40 52.2 1785 2805 11540 11557 50.4 50 5305 11727 11707 51.1 42.9 0.7 188 73.1 41 40 51.4 1786 2806 98 118 50.6 42.9 5306 642 622 51.6 47.6 0.9 545 79.1 49.7 40 55.6 1787 2807 15211 15230 50.2 45 5307 15595 15576 50.8 45 0.6 385 75.1 41.3 40 52.7 1788 2808 15255 15273 50.3 52.6 5308 15767 15747 50 42.9 0.3 513 75.1 40.2 40 52.6 1789 2809 12373 12391 50.8 47.4 5309 12911 12891 51.2 47.6 0.4 539 76.1 42.5 40 53.6 1790 2810 18009 18028 51.6 55 5310 18697 18679 51.9 52.6 0.2 689 76.4 42.5 40 54 1791 2811 18009 18028 51.6 55 5311 18696 18673 53.4 41.7 1.8 688 76.3 42.4 40 54 1792 2812 18009 18028 51.6 55 5312 18239 18220 50 45 1.6 231 74.9 43.7 40 52.5 1793 2813 18009 18028 51.6 55 5313 18238 18219 50.3 45 1.4 230 75 43.9 40 52.7 1794 2814 24417 24436 52.6 50 5314 25079 25061 52.7 52.6 0.1 663 75.8 41.3 40 54 1795 2815 10250 10271 50.6 45.5 5315 10356 10336 52.4 47.6 1.8 107 70.8 42.1 40 49.9 1796 2816 1356 1375 53.8 55 5316 1484 1464 54.3 47.6 0.5 129 74.4 48.1 40 53.3 1797 2817 1356 1375 53.8 55 5317 1484 1465 53.8 50 0 129 74.4 48.1 40 53.3 1798 2818 1356 1375 53.8 55 5318 1484 1466 53.1 52.6 0.7 129 74.4 48.1 40 53.1 1799 2819 24418 24436 50 47.4 5319 24560 24542 50.2 47.4 0.1 143 72.4 42 40 50.8 1800 2820 18008 18028 53 52.4 5320 18696 18672 53.9 40 0.9 689 76.3 42.4 40 54.4 1801 2821 25363 25381 51.1 52.6 5321 25646 25627 50.5 45 0.7 284 74.1 40.5 40 52.1 1802 2822 9131 9151 50.4 42.9 5322 9333 9315 52.2 52.6 1.9 203 74.6 43.8 40 52.4 1803 2823 9131 9151 50.4 42.9 5323 9374 9353 50.1 40.9 0.3 244 74.6 42.6 40 52.4 1804 2824 24380 24399 55 55 5324 24582 24560 54.2 52.2 0.9 203 74.4 43.3 40 53.4 1805 2825 19802 19820 53 52.6 5325 19921 19900 51.8 45.5 1.2 120 72.4 44.2 40 51.3 1806 2826 3055 3075 51.8 47.6 5326 3210 3190 50.5 47.6 1.3 156 74.2 45.5 40 52.2 1807 2827 3055 3075 51.8 47.6 5327 3207 3187 50.5 47.6 1.3 153 74 45.1 40 52 1808 2828 3055 3076 52.4 45.5 5328 3210 3190 50.5 47.6 2 156 74.2 45.5 40 52.2 1809 2829 24379 24398 55 55 5329 24582 24560 54.2 52.2 0.9 204 74.3 43.1 40 53.3 1810 2830 7876 7895 51.5 45 5330 8054 8035 50.4 50 1.1 179 73.5 42.5 40 51.7 1811 2831 3055 3076 52.4 45.5 5331 3207 3187 50.5 47.6 2 153 74 45.1 40 52 1812 2832 9130 9150 51.3 42.9 5332 9324 9300 52.9 40 1.6 195 73.9 42.6 40 52.2 1813 2833 9130 9150 51.3 42.9 5333 9324 9301 52.4 41.7 1.1 195 73.9 42.6 40 52.2 1814 2834 8794 8813 51.6 45 5334 9324 9301 52.4 41.7 0.8 531 75.7 41.6 40 53.6 1815 2835 8794 8813 51.6 45 5335 9324 9300 52.9 40 1.3 531 75.7 41.6 40 53.6 1816 2836 9130 9150 51.3 42.9 5336 9328 9310 51 52.6 0.3 199 74.2 43.2 40 52.4 1817 2837 9130 9150 51.3 42.9 5337 9333 9315 52.2 52.6 0.9 204 74.5 43.6 40 52.6 1818 2838 24179 24200 53.3 40.9 5338 24807 24786 51.7 45.5 1.6 629 75.8 41.3 40 53.7 1819 2839 4593 4613 51.5 47.6 5339 4708 4690 50.3 47.4 1.3 116 71.4 42.2 40 50.2 1820 2840 9130 9150 51.3 42.9 5340 9374 9353 50.1 40.9 1.2 245 74.6 42.4 40 52.3 1821 2841 29180 29199 50.1 40 5341 29412 29393 50.3 45 0.2 233 75.5 45.1 40 53 1822 2842 25348 25366 51.2 47.4 5342 25772 25753 51.9 50 0.8 425 74.9 40.5 40 52.9 1823 2843 24179 24200 53.3 40.9 5343 24815 24792 53.4 41.7 0.2 637 75.8 41.3 40 54.1 1824 2844 8794 8813 51.6 45 5344 9101 9081 50.5 47.6 1.2 308 74.8 41.6 40 52.6 1825 2845 16861 16880 50.8 50 5345 17056 17035 51.8 45.5 1 196 74.7 44.4 40 52.6 1826 2846 16562 16581 52.6 50 5346 17038 17021 50.7 50 1.9 477 75.7 41.9 40 53.3 1827 2847 16562 16581 52.6 50 5347 17039 17022 51.4 50 1.2 478 75.6 41.8 40 53.5 1828 2848 16562 16581 52.6 50 5348 17041 17023 53.5 52.6 0.9 480 75.7 41.9 40 53.8 1829 2849 3090 3110 50.3 42.9 5349 3647 3628 50.6 45 0.3 558 76.2 42.7 40 53.5 1830 2850 16562 16580 51.9 52.6 5350 17038 17021 50.7 50 1.2 477 75.7 41.9 40 53.3 1831 2851 16562 16580 51.9 52.6 5351 17039 17022 51.4 50 0.5 478 75.6 41.8 40 53.5 1832 2852 24178 24198 52.7 42.9 5352 24815 24792 53.4 41.7 0.7 638 75.7 41.2 40 53.9 1833 2853 16562 16580 51.9 52.6 5353 17041 17023 53.5 52.6 1.6 480 75.7 41.9 40 53.6 1834 2854 24179 24198 51 45 5354 24807 24786 51.7 45.5 0.7 629 75.8 41.3 40 53.5 1835 2855 24179 24198 51 45 5355 24818 24797 51.6 40.9 0.6 640 75.8 41.2 40 53.4 1836 2856 3090 3110 50.3 42.9 5356 3646 3625 52 40.9 1.7 557 76.1 42.5 40 53.5 1837 2857 3089 3110 51.8 45.5 5357 3650 3631 53.1 50 1.3 562 76.3 42.9 40 54 1838 2858 29259 29279 54 52.4 5358 29358 29339 52.8 50 1.1 100 72.4 47 40 51.6 1839 2859 8794 8813 51.6 45 5359 8928 8911 51.9 50 0.2 135 72.2 42.2 40 51.1 1840 2860 24176 24197 52.1 40.9 5360 24815 24792 53.4 41.7 1.3 640 75.8 41.2 40 53.8 1841 2861 29259 29277 50.9 52.6 5361 29358 29339 52.8 50 2 100 72.4 47 40 51.1 1842 2862 29257 29276 51.3 50 5362 29358 29339 52.8 50 1.5 102 72.6 47.1 40 51.3 1843 2863 9915 9935 51.8 47.6 5363 10017 9999 52.8 52.6 1 103 72.9 47.6 40 51.6 1844 2864 4639 4659 51.1 47.6 5364 5306 5288 52.4 52.6 1.3 668 75.6 40.9 40 53.4 1845 2865 24178 24197 50.3 40 5365 24807 24786 51.7 45.5 1.4 630 75.8 41.3 40 53.2 1846 2866 28653 28671 50.2 52.6 5366 29414 29395 50.5 50 0.3 762 78 46.2 40 54.7 1847 2867 28653 28671 50.2 52.6 5367 29412 29393 50.3 45 0.1 760 78 46.2 40 54.7 1848 2868 28652 28671 52.8 55 5368 29358 29339 52.8 50 0 707 78 46.4 40 55.5 1849 2869 15752 15772 50.8 47.6 5369 16213 16195 50.8 52.6 0 462 75.4 41.3 40 53.1 1850 2870 24178 24197 50.3 40 5370 24818 24797 51.6 40.9 1.4 641 75.7 41.2 40 53.2 1851 2871 19794 19814 51.7 47.6 5371 19909 19885 52.5 40 0.8 116 71.8 43.1 40 50.8 1852 2872 8866 8885 51.1 45 5372 9341 9322 51.1 50 0 476 75.6 41.8 40 53.4 1853 2873 15951 15973 52.1 43.5 5373 16175 16155 51.8 47.6 0.3 225 73.7 40.9 40 52.2 1854 2874 24174 24195 52.5 40.9 5374 24815 24791 54.5 40 2 642 75.8 41.3 40 53.9 1855 2875 8866 8885 51.1 45 5375 9340 9319 50.8 45.5 0.3 475 75.6 41.7 40 53.2 1856 2876 15951 15973 52.1 43.5 5376 16169 16151 51.3 52.6 0.8 219 73.5 40.6 40 51.9 1857 2877 15951 15974 53.3 41.7 5377 16175 16154 53.4 45.5 0.1 225 73.7 40.9 40 52.7 1858 2878 27437 27456 50.2 40 5378 27541 27521 51.7 47.6 1.5 105 71.8 44.8 40 50.4 1859 2879 15650 15674 52.9 40 5379 16210 16192 54.3 52.6 1.4 561 75.1 40.1 40 53.6 1860 2880 8866 8885 51.1 45 5380 9334 9316 51.3 52.6 0.2 469 75.7 42 40 53.4 1861 2881 8866 8885 51.1 45 5381 9310 9291 51.2 45 0.1 445 75.3 41.3 40 53.2 1862 2882 8866 8885 51.1 45 5382 9252 9234 51.4 52.6 0.3 387 75.1 41.3 40 53 1863 2883 3360 3379 50.7 45 5383 3494 3473 50.4 40.9 0.3 135 73.7 45.9 40 51.8 1864 2884 8866 8885 51.1 45 5384 9248 9229 50.1 45 1 383 75.1 41.3 40 52.7 1865 2885 18081 18099 51.2 52.6 5385 18697 18679 51.9 52.6 0.7 617 76.3 42.6 40 53.9 1866 2886 8865 8884 50.4 45 5386 9257 9238 50.5 45 0.1 393 75 41 40 52.7 1867 2887 18081 18099 51.2 52.6 5387 18239 18220 50 45 1.2 159 74 44.7 40 51.9 1868 2888 18081 18099 51.2 52.6 5388 18238 18219 50.3 45 0.9 158 74.1 44.9 40 52 1869 2889 28117 28135 50.6 52.6 5389 28505 28487 50.2 47.4 0.4 389 79.5 51.9 40 55.8 1870 2890 8866 8885 51.1 45 5390 9109 9087 50.5 43.5 0.6 244 73.9 41 40 52 1871 2891 9055 9079 52.8 40 5391 9724 9706 51.3 52.6 1.5 670 75.4 40.3 40 53.3 1872 2892 3403 3423 54.1 47.6 5392 3502 3478 55.8 48 1.7 100 71.6 45 40 51.5 1873 2893 28855 28874 52.9 50 5393 29306 29288 53.5 52.6 0.6 452 77.1 45.6 40 54.9 1874 2894 24173 24194 52.5 40.9 5394 24815 24792 53.4 41.7 0.9 643 75.8 41.2 40 53.9 1875 2895 3094 3113 50 50 5395 3647 3628 50.6 45 0.6 554 76.2 42.8 40 53.5 1876 2896 24174 24194 50.9 42.9 5396 24807 24786 51.7 45.5 0.8 634 75.8 41.3 40 53.4 1877 2897 28856 28875 52.2 50 5397 29306 29288 53.5 52.6 1.3 451 77.1 45.7 40 54.7 1878 2898 24174 24194 50.9 42.9 5398 24818 24797 51.6 40.9 0.7 645 75.8 41.2 40 53.4 1879 2899 28857 28876 51.7 45 5399 29306 29288 53.5 52.6 1.8 450 77.1 45.6 40 54.6 1880 2900 8858 8877 51.2 45 5400 9254 9236 50.6 47.4 0.6 397 75 41.1 40 52.8 1881 2901 16553 16571 53.4 52.6 5401 16777 16758 51.5 50 1.9 225 73.7 40.9 40 52.1 1882 2902 29197 29219 54.8 47.8 5402 29301 29282 55.3 55 0.5 105 73.4 48.6 40 52.9 1883 2903 29198 29219 52.6 45.5 5403 29306 29288 53.5 52.6 0.9 109 73.3 47.7 40 52.2 1884 2904 28857 28877 52.3 42.9 5404 29306 29288 53.5 52.6 1.2 450 77.1 45.6 40 54.8 1885 2905 29199 29219 51.2 42.9 5405 29298 29280 51.4 52.6 0.2 100 72.4 47 40 51.1 1886 2906 3094 3113 50 50 5406 3646 3625 52 40.9 2 553 76.2 42.7 40 53.4 1887 2907 3224 3243 52.3 50 5407 3650 3631 53.1 50 0.8 427 75.5 41.9 40 53.7 1888 2908 29195 29216 53.8 45.5 5408 29306 29287 54.6 55 0.8 112 73.6 48.2 40 52.8 1889 2909 28867 28885 51.5 52.6 5409 29358 29339 52.8 50 1.4 492 76.9 44.9 40 54.4 1890 2910 29196 29216 52.5 47.6 5410 29298 29279 52.6 55 0.1 103 73.3 48.5 40 52.1 1891 2911 28867 28886 53.2 50 5411 29415 29395 53.4 52.4 0.2 549 77.1 45 40 55 1892 2912 3093 3113 51.7 47.6 5412 3650 3631 53.1 50 1.4 558 76.3 42.8 40 54 1893 2913 3225 3243 50.9 52.6 5413 3646 3625 52 40.9 1.2 422 75.4 41.7 40 53.1 1894 2914 3225 3243 50.9 52.6 5414 3647 3628 50.6 45 0.3 423 75.5 41.8 40 53.1 1895 2915 28867 28886 53.2 50 5415 29306 29287 54.6 55 1.4 440 76.9 45.2 40 54.9 1896 2916 3223 3241 50.2 52.6 5416 3500 3481 51.2 50 1 278 74.7 42.1 40 52.5 1897 2917 28867 28886 53.2 50 5417 29298 29279 52.6 55 0.5 432 76.8 45.1 40 54.7 1898 2918 24034 24053 53.4 55 5418 24815 24791 54.5 40 1.1 782 76.3 42.1 40 54.5 1899 2919 3221 3239 51.5 52.6 5419 3650 3631 53.1 50 1.6 430 75.5 41.9 40 53.4 1900 2920 18080 18099 53 50 5420 18696 18673 53.4 41.7 0.5 617 76.2 42.5 40 54.3 1901 2921 3095 3116 51.9 45.5 5421 3650 3631 53.1 50 1.2 556 76.2 42.8 40 54 1902 2922 18080 18099 53 50 5422 18696 18672 53.9 40 1 617 76.2 42.5 40 54.3 1903 2923 28868 28887 50.7 45 5423 29298 29279 52.6 55 1.9 431 76.8 45 40 54.1 1904 2924 3218 3238 52.1 47.6 5424 3650 3631 53.1 50 1 433 75.5 41.8 40 53.6 1905 2925 8867 8886 50.7 50 5425 9252 9234 51.4 52.6 0.8 386 75.1 41.5 40 52.9 1906 2926 28867 28887 53.7 47.6 5426 29306 29287 54.6 55 0.8 440 76.9 45.2 40 55.1 1907 2927 3218 3237 50.5 45 5427 3497 3478 51.3 50 0.8 280 74.6 41.8 40 52.5 1908 2928 29195 29215 53.2 47.6 5428 29306 29287 54.6 55 1.4 112 73.6 48.2 40 52.6 1909 2929 29196 29215 51.8 50 5429 29298 29279 52.6 55 0.8 103 73.3 48.5 40 51.9 1910 2930 3218 3237 50.5 45 5430 3500 3481 51.2 50 0.6 283 74.6 41.7 40 52.5 1911 2931 8867 8886 50.7 50 5431 9245 9226 50 45 0.6 379 75 41.2 40 52.6 1912 2932 28868 28888 51.4 42.9 5432 29298 29279 52.6 55 1.2 431 76.8 45 40 54.3 1913 2933 8867 8886 50.7 50 5433 9107 9086 51.6 45.5 0.9 241 74.1 41.5 40 52.2 1914 2934 28867 28888 54.3 45.5 5434 29306 29287 54.6 55 0.3 440 76.9 45.2 40 55.2 1915 2935 19906 19925 50.1 50 5435 20615 20597 50.6 47.4 0.5 710 75.5 40.3 40 53 1916 2936 16551 16568 51.1 50 5436 16775 16756 50.3 45 0.8 225 73.8 41.3 40 51.9 1917 2937 8861 8880 50.2 45 5437 9341 9322 51.1 50 0.9 481 75.5 41.6 40 53 1918 2938 16368 16387 50.2 45 5438 16781 16761 51.3 47.6 1 414 75 40.8 40 52.7 1919 2939 3055 3074 51.1 50 5439 3209 3189 50.5 47.6 0.6 155 74.1 45.2 40 52.1 1920 2940 3217 3236 51.1 50 5440 3650 3631 53.1 50 2 434 75.5 41.9 40 53.3 1921 2941 28868 28889 52 40.9 5441 29298 29279 52.6 55 0.6 431 76.8 45 40 54.5 1922 2942 28867 28889 54.8 43.5 5442 29306 29287 54.6 55 0.2 440 76.9 45.2 40 55.3 1923 2943 3404 3422 50.5 47.4 5443 3503 3484 51.5 50 0.9 100 71.6 45 40 50.4 1924 2944 16368 16387 50.2 45 5444 16777 16758 51.5 50 1.2 410 75 40.7 40 52.6 1925 2945 24029 24047 52.1 52.6 5445 24815 24792 53.4 41.7 1.3 787 76.3 42.1 40 54.1 1926 2946 16368 16387 50.2 45 5446 16711 16691 51 42.9 0.8 344 75.1 41.9 40 52.7 1927 2947 28867 28890 55.2 41.7 5447 29306 29287 54.6 55 0.6 440 76.9 45.2 40 55.3 1928 2948 29196 29214 51.1 52.6 5448 29298 29279 52.6 55 1.5 103 73.3 48.5 40 51.7 1929 2949 18488 18507 53.7 55 5449 19224 19200 52.4 40 1.3 737 76 41.5 40 54 1930 2950 28395 28413 50.2 42.1 5450 28506 28488 50.2 47.4 0 112 74.4 50 40 52.2 1931 2951 16551 16568 51.1 50 5451 17032 17011 52 45.5 0.9 482 75.8 42.1 40 53.5 1932 2952 28871 28891 50.9 42.9 5452 29358 29339 52.8 50 1.9 488 76.9 44.9 40 54.2 1933 2953 28871 28891 50.9 42.9 5453 29298 29280 51.4 52.6 0.5 428 76.8 45.1 40 54.2 1934 2954 28870 28891 52.2 40.9 5454 29306 29288 53.5 52.6 1.2 437 76.8 45.1 40 54.6 1935 2955 28868 28891 53.8 41.7 5455 29301 29282 55.3 55 1.5 434 76.9 45.2 40 55.1 1936 2956 3404 3422 50.5 47.4 5456 3504 3485 50.4 45 0.1 101 71.5 44.6 40 50.3 1937 2957 29195 29213 51.9 52.6 5457 29298 29279 52.6 55 0.7 104 73.1 48.1 40 51.9 1938 2958 28938 28956 50.8 47.4 5458 29298 29280 51.4 52.6 0.6 361 76.4 44.9 40 53.8 1939 2959 18488 18507 53.7 55 5459 19210 19191 52 50 1.7 723 76 41.6 40 53.9 1940 2960 3095 3116 51.9 45.5 5460 3647 3628 50.6 45 1.3 553 76.2 42.7 40 53.6 1941 2961 3214 3233 51.1 50 5461 3497 3478 51.3 50 0.2 284 74.7 41.9 40 52.7 1942 2962 24017 24039 53 43.5 5462 24815 24791 54.5 40 1.5 799 76.2 41.9 40 54.4 1943 2963 3095 3116 51.9 45.5 5463 3646 3625 52 40.9 0.1 552 76.1 42.6 40 54 1944 2964 18550 18571 50.4 40.9 5464 19215 19194 50.2 40.9 0.2 666 75.8 41.1 40 53.2 1945 2965 3214 3233 51.1 50 5465 3500 3481 51.2 50 0.1 287 74.7 41.8 40 52.7 1946 2966 18586 18603 50.4 44.4 5466 19224 19200 52.4 40 1.9 639 75.6 40.8 40 53.1 1947 2967 18586 18603 50.4 44.4 5467 19217 19196 50.2 40.9 0.2 632 75.6 41 40 53.1 1948 2968 18586 18603 50.4 44.4 5468 19215 19194 50.2 40.9 0.2 630 75.6 41 40 53.1 1949 2969 18590 18608 50.6 42.1 5469 19224 19200 52.4 40 1.8 635 75.6 40.9 40 53.2 1950 2970 15255 15273 50.3 52.6 5470 15767 15746 50.7 40.9 0.4 513 75.1 40.2 40 52.7 1951 2971 28942 28961 50.2 45 5471 29414 29395 50.5 50 0.3 473 76.8 44.6 40 53.9 1952 2972 18590 18608 50.6 42.1 5472 19217 19196 50.2 40.9 0.3 628 75.7 41.1 40 53.1 1953 2973 3055 3074 51.1 50 5473 3207 3187 50.5 47.6 0.6 153 74 45.1 40 52 1954 2974 18590 18608 50.6 42.1 5474 19215 19194 50.2 40.9 0.3 626 75.7 41.1 40 53.1 1955 2975 18591 18611 51.7 42.9 5475 19224 19200 52.4 40 0.7 634 75.7 41 40 53.6 1956 2976 18591 18611 51.7 42.9 5476 19217 19196 50.2 40.9 1.4 627 75.7 41.1 40 53.2 1957 2977 18591 18611 51.7 42.9 5477 19215 19194 50.2 40.9 1.4 625 75.7 41.1 40 53.2 1958 2978 28546 28565 52.2 50 5478 28672 28654 50.6 52.6 1.6 127 76.5 53.5 40 53.8 1959 2979 29191 29210 54.4 55 5479 29415 29395 53.4 52.4 1 225 75.7 45.8 40 54.1 1960 2980 7880 7900 50.3 42.9 5480 8190 8172 50.3 47.4 0 311 74.9 41.8 40 52.6 1961 2981 3167 3189 51.6 43.5 5481 3650 3631 53.1 50 1.5 484 75.8 42.1 40 53.6 1962 2982 28965 28984 52.9 55 5482 29306 29288 53.5 52.6 0.6 342 76.5 45.3 40 54.5 1963 2983 3166 3188 51.6 43.5 5483 3650 3631 53.1 50 1.5 485 75.8 42.3 40 53.7 1964 2984 8867 8887 52.3 47.6 5484 9101 9081 50.5 47.6 1.9 235 74.1 41.7 40 52.1 1965 2985 23843 23863 50.3 42.9 5485 24013 23995 50.3 47.4 0 171 73.7 43.3 40 51.8 1966 2986 3403 3421 53.1 52.6 5486 3503 3484 51.5 50 1.7 101 71.9 45.5 40 50.9 1967 2987 16549 16567 54.9 52.6 5487 16777 16756 53.4 45.5 1.5 229 74 41.5 40 52.9 1968 2988 8868 8889 50.4 40.9 5488 9109 9087 50.5 43.5 0.1 242 73.9 40.9 40 51.9 1969 2989 8861 8880 50.2 45 5489 9311 9292 50.7 50 0.6 451 75.3 41.2 40 52.9 1970 2990 8868 8889 50.4 40.9 5490 9257 9238 50.5 45 0.1 390 75 41 40 52.7 1971 2991 8868 8889 50.4 40.9 5491 9313 9294 50.4 50 0 446 75.4 41.5 40 53 1972 2992 23841 23859 50.5 52.6 5492 24013 23995 50.3 47.4 0.1 173 74 43.9 40 52 1973 2993 28548 28568 50.5 42.9 5493 28672 28654 50.6 52.6 0 125 76.2 52.8 40 53.6 1974 2994 8867 8888 52.7 45.5 5494 9310 9291 51.2 45 1.5 444 75.4 41.4 40 53.2 1975 2995 28968 28988 50.9 47.6 5495 29298 29279 52.6 55 1.8 331 76.2 44.7 40 53.7 1976 2996 19907 19926 52.1 55 5496 20615 20597 50.6 47.4 1.6 709 75.5 40.3 40 53.1 1977 2997 8861 8880 50.2 45 5497 9252 9235 50.1 50 0.1 392 75 41.1 40 52.6 1978 2998 19909 19929 50.7 52.4 5498 20615 20597 50.6 47.4 0.2 707 75.5 40.3 40 53.1 1979 2999 3361 3382 51.9 45.5 5499 3500 3481 51.2 50 0.7 140 74.1 46.4 40 52.3 1980 3000 18696 18715 51.7 50 5500 18881 18862 50.2 45 1.5 186 74.1 43.5 40 52.1 1981 3001 28968 28989 51.5 45.5 5501 29298 29279 52.6 55 1.1 331 76.2 44.7 40 53.9 1982 3002 19709 19730 51.3 40.9 5502 19923 19903 50.9 47.6 0.4 215 73.9 41.9 40 52.1 1983 3003 3361 3382 51.9 45.5 5503 3497 3478 51.3 50 0.6 137 74.1 46.7 40 52.4 1984 3004 3361 3384 53.7 41.7 5504 3495 3473 51.8 43.5 1.9 135 74 46.7 40 52.5 1985 3005 19709 19730 51.3 40.9 5505 19924 19905 50.1 50 1.2 216 73.9 41.7 40 51.8 1986 3006 16378 16397 50.4 45 5506 16711 16691 51 42.9 0.6 334 75.2 42.2 40 52.9 1987 3007 3361 3382 51.9 45.5 5507 3504 3485 50.4 45 1.5 144 74.3 46.5 40 52.2 1988 3008 18704 18724 50.8 47.6 5508 19406 19388 50.6 47.4 0.1 703 75.4 40.3 40 53.1 1989 3009 8868 8889 50.4 40.9 5509 9314 9295 51.1 50 0.7 447 75.5 41.6 40 53 1990 3010 3361 3382 51.9 45.5 5510 3503 3484 51.5 50 0.5 143 74.4 46.9 40 52.6 1991 3011 19709 19730 51.3 40.9 5511 19931 19912 50.9 55 0.4 223 74.2 42.2 40 52.3 1992 3012 16548 16566 54.9 52.6 5512 16777 16756 53.4 45.5 1.5 230 73.9 41.3 40 52.9 1993 3013 8868 8889 50.4 40.9 5513 9315 9296 50 45 0.4 448 75.4 41.5 40 52.9 1994 3014 22321 22341 51.6 42.9 5514 22460 22441 50.7 45 0.9 140 71.5 40 40 50.3 1995 3015 29182 29202 51.2 42.9 5515 29412 29393 50.3 45 0.9 231 75.4 45 40 53 1996 3016 22173 22193 51 42.9 5516 22460 22441 50.7 45 0.3 288 74.1 40.3 40 52.1 1997 3017 29181 29201 52.4 47.6 5517 29413 29393 51.1 42.9 1.3 233 75.5 45.1 40 53.3 1998 3018 18704 18724 50.8 47.6 5518 18881 18862 50.2 45 0.5 178 73.8 43.3 40 51.9 1999 3019 20751 20771 51.3 47.6 5519 21301 21278 51.3 41.7 0 551 75.5 41 40 53.3 2000 3020 29181 29200 50 45 5520 29412 29393 50.3 45 0.3 232 75.5 45.3 40 53 2001 3021 20751 20771 51.3 47.6 5521 21304 21283 50.5 40.9 0.8 554 75.5 41 40 53.1 2002 3022 29173 29197 54.2 40 5522 29415 29395 53.4 52.4 0.8 243 75.7 45.3 40 54.1 2003 3023 8867 8888 52.7 45.5 5523 9247 9226 52 45.5 0.7 381 75 41.2 40 53.2 2004 3024 8867 8888 52.7 45.5 5524 9255 9236 51.1 45 1.6 389 75 41.1 40 52.9 2005 3025 29178 29198 51.4 42.9 5525 29412 29393 50.3 45 1.1 235 75.5 45.1 40 53.1 2006 3026 3163 3185 53.6 47.8 5526 3650 3631 53.1 50 0.5 488 75.9 42.4 40 54.2 2007 3027 19800 19817 50.4 50 5527 20033 20016 50.4 50 0 234 74.9 43.6 41 52.7 2008 3028 8867 8886 50.7 50 5528 9376 9355 51 40.9 0.3 510 75.7 41.8 41 53.3 2009 3029 19800 19817 50.4 50 5529 19930 19910 50.6 47.6 0.2 131 72.6 43.5 41 51 2010 3030 24418 24439 52.9 45.5 5530 25082 25064 51.1 52.6 1.8 665 75.8 41.2 41 53.5 2011 3031 25771 25790 51.1 45 5531 26182 26161 51.2 40.9 0.1 412 74.8 40.3 41 52.8 2012 3032 12976 12994 50.3 47.4 5532 13326 13306 50.7 42.9 0.3 351 76.1 44.2 41 53.5 2013 3033 12976 12994 50.3 47.4 5533 13328 13307 51.2 45.5 0.9 353 76.1 44.2 41 53.5 2014 3034 2823 2844 50.4 45.5 5534 3500 3481 51.2 50 0.7 678 76.3 42.5 41 53.7 2015 3035 18009 18028 51.6 55 5535 18223 18205 53.3 52.6 1.7 215 74.5 43.3 41 52.7 2016 3036 8223 8240 50.4 50 5536 8933 8916 52.2 50 1.8 711 75.4 40.1 41 53 2017 3037 29180 29199 50.1 40 5537 29414 29395 50.5 50 0.4 235 75.5 45.1 41 53 2018 3038 19800 19817 50.4 50 5538 19925 19906 50.1 50 0.4 126 72.4 43.7 41 50.8 2019 3039 25772 25793 52.4 40.9 5539 26183 26162 52.8 45.5 0.4 412 74.8 40.3 41 53.2 2020 3040 14951 14975 52.2 40 5540 15152 15135 51.4 50 0.8 202 73.4 41.1 41 51.9 2021 3041 2823 2844 50.4 45.5 5541 3503 3484 51.5 50 1 681 76.4 42.6 41 53.7 2022 3042 18075 18095 50.6 47.6 5542 18231 18210 52.2 45.5 1.6 157 73.6 43.9 41 51.8 2023 3043 5 23 51.3 52.6 5543 269 251 51.1 52.6 0.1 265 76.4 46.4 41 53.9 2024 3044 9140 9159 50.1 45 5544 9249 9231 50.8 47.4 0.7 110 71.3 42.7 41 50 2025 3045 24418 24439 52.9 45.5 5545 24815 24791 54.5 40 1.6 398 75.9 43.2 41 54.1 2026 3046 8794 8813 51.6 45 5546 9358 9338 51 42.9 0.6 565 75.8 41.8 41 53.5 2027 3047 24418 24439 52.9 45.5 5547 24807 24786 51.7 45.5 1.2 390 75.9 43.3 41 53.8 2028 3048 2387 2405 51.6 52.6 5548 3186 3165 50.4 40.9 1.2 800 76.9 43.5 41 54.1 2029 3049 24418 24439 52.9 45.5 5549 24527 24506 51.7 40.9 1.2 110 71.3 42.7 41 50.5 2030 3050 24418 24439 52.9 45.5 5550 24517 24494 53.2 41.7 0.3 100 70.8 43 41 50.5 2031 3051 4255 4276 51.7 45.5 5551 4836 4817 51.2 45 0.5 582 75.9 41.9 41 53.6 2032 3052 24420 24440 50.8 42.9 5552 25082 25064 51.1 52.6 0.3 663 75.7 41 41 53.3 2033 3053 8867 8887 52.3 47.6 5553 9250 9232 51.6 47.4 0.8 384 75.1 41.4 41 53.2 2034 3054 14951 14975 52.2 40 5554 15275 15257 50.8 52.6 1.3 325 74.6 40.9 41 52.6 2035 3055 2387 2405 51.6 52.6 5555 3185 3164 51 45.5 0.7 799 76.9 43.6 41 54.2 2036 3056 8865 8884 50.4 45 5556 9252 9235 50.1 50 0.3 388 75.1 41.2 41 52.7 2037 3057 24420 24440 50.8 42.9 5557 24818 24797 51.6 40.9 0.8 399 75.8 42.9 41 53.4 2038 3058 24420 24440 50.8 42.9 5558 24807 24786 51.7 45.5 0.9 388 75.8 43 41 53.4 2039 3059 11541 11560 50.1 45 5559 12110 12090 51.1 42.9 1 570 75.9 41.9 41 53.3 2040 3060 2387 2405 51.6 52.6 5560 2672 2653 51.6 50 0 286 77 47.6 41 54.5 2041 3061 24420 24440 50.8 42.9 5561 24526 24506 50.3 42.9 0.5 107 70.8 42.1 41 49.8 2042 3062 11540 11557 50.4 50 5562 12110 12090 51.1 42.9 0.7 571 76 42 41 53.4 2043 3063 6263 6282 50.9 45 5563 6483 8463 50.2 42.9 0.7 221 73.7 41.2 41 51.8 2044 3064 24418 24440 55 47.8 5564 24815 24791 54.5 40 0.5 398 75.9 43.2 41 54.6 2045 3065 18075 118095 50.6 47.6 5565 18233 18214 52 50 1.4 159 74 44.7 41 52.1 2046 3066 18075 18095 50.6 47.6 5566 18233 18215 51.3 52.6 0.7 159 74 44.7 41 52.1 2047 3067 2429 2447 50.2 47.4 5567 3055 3036 50.6 50 0.4 627 76.3 42.6 41 53.6 2048 3068 19800 19818 52.1 52.6 5568 19917 19896 50.9 45.5 1.2 118 71.9 43.2 41 50.7 2049 3069 24481 24500 50.1 45 5569 24936 24919 51.8 50 1.7 456 75.7 42.1 41 53.1 2050 3070 276 294 50.5 47.4 5570 713 695 50.7 47.4 0.2 438 79.1 50.7 41 55.7 2051 3071 19801 19819 53.2 52.6 5571 19927 19908 52.1 55 1.1 127 72.7 44.1 41 51.6 2052 3072 19801 19819 53.2 52.6 5572 19925 19905 51.4 52.4 1.9 125 72.5 44 41 51.3 2053 3073 3800 3824 53.6 40 5573 4318 4294 54.4 40 0.8 519 75.3 40.8 41 53.9 2054 3074 11540 11557 50.4 50 5574 12258 12238 50.3 42.9 0.2 719 76.2 42 41 53.5 2055 3075 24482 24502 50.3 42.9 5575 24938 24921 50.4 50 0.1 457 75.6 41.8 41 53.1 2056 3076 24482 24502 50.3 42.9 5576 24807 24786 51.7 45.5 1.4 326 75.4 42.9 41 53 2057 3077 8867 8888 52.7 45.5 5577 9364 9346 53.9 52.6 1.2 498 75.8 42.2 41 54 2058 3078 24481 24502 51.5 45.5 5578 25080 25062 53.5 52.6 2 600 75.5 40.8 41 53.4 2059 3079 8865 8884 50.4 45 5579 9107 9086 51.6 45.5 1.2 243 74 41.2 41 52 2060 3080 8867 8888 52.7 45.5 5580 9313 9293 52.1 47.6 0.6 447 75.5 41.6 41 53.5 2061 3081 2427 2445 52.1 52.6 5581 3055 3036 50.6 50 1.5 629 76.4 42.8 41 53.7 2062 3082 2823 2844 50.4 45.5 5582 3504 3485 50.4 45 0.1 682 76.3 42.5 41 53.7 2063 3083 24483 24503 51 42.9 5583 25085 25068 50.3 50 0.6 603 75.4 40.6 41 53 2064 3084 15255 15273 50.3 52.6 5584 15649 15632 50.1 50 0.2 395 75.1 41.3 41 52.7 2065 3085 24483 24503 51 42.9 5585 25082 25064 51.1 52.6 0.1 600 75.5 40.8 41 53.3 2066 3086 8867 8886 50.7 50 5586 9375 9354 50.4 40.9 0.3 509 75.7 41.8 41 53.2 2067 3087 12976 12994 50.3 47.4 5587 13329 13308 50.5 40.9 0.2 354 76.1 44.1 41 53.4 2068 3088 24483 24503 51 42.9 5588 25081 25063 52.4 52.6 1.4 599 75.5 40.7 41 53.2 2069 3089 379 398 50.1 45 5589 941 922 50.5 50 0.4 563 78.7 48.8 41 55.2 2070 3090 24483 24503 51 42.9 5590 24936 24919 51.8 50 0.8 454 75.7 42.1 41 53.4 2071 3091 19802 19820 53 52.6 5591 19927 19908 52.1 55 0.8 126 72.8 44.4 41 51.7 2072 3092 9934 9953 50.7 50 5592 10670 10649 51.3 40.9 0.6 737 75.7 40.8 41 53.3 2073 3093 8866 8885 51.1 45 5593 9312 9293 50.6 45 0.5 447 75.4 41.4 41 53 2074 3094 19846 19866 51.2 42.9 5594 20033 20016 50.4 50 0.8 188 74.2 43.6 41 52.2 2075 3095 19848 19867 50.7 45 5595 20033 20016 50.4 50 0.3 186 74.1 43.5 41 52.1 2076 3096 9538 9558 50.9 42.9 5596 10017 9999 52.8 52.6 1.9 480 75.5 41.5 41 53.2 2077 3097 8220 8238 51.5 47.4 5597 8933 8916 52.2 50 0.7 714 75.4 40.1 41 53.3 2078 3098 9140 9159 50.1 45 5598 9249 9232 50 50 0.1 110 71.3 42.7 41 50 2079 3099 12976 12994 50.3 47.4 5599 13332 13312 50.9 47.6 0.6 357 76.2 44.3 41 53.5 2080 3100 15752 15772 50.8 47.6 5600 16174 16154 50.4 42.9 0.4 423 75.1 40.9 41 52.8 2081 3101 18074 18094 51.1 42.9 5601 18232 18212 50.6 47.6 0.5 159 73.7 44 41 51.9 2082 3102 24559 24579 52 52.4 5602 25081 25063 52.4 52.6 0.4 523 75.5 41.1 41 53.5 2083 3103 24559 24579 52 52.4 5603 25079 25061 52.7 52.6 0.7 521 75.5 41.3 41 53.6 2084 3104 3169 3191 52.1 47.8 5604 3650 3631 53.1 50 1 482 75.9 42.3 41 53.8 2085 3105 28117 28135 50.6 52.6 5605 28672 28654 50.6 52.6 0.1 556 80 52 41 56.3 2086 3106 1809 1829 50.6 42.9 5606 2103 2082 52 45.5 1.5 295 75.4 43.4 41 53 2087 3107 24559 24579 52 52.4 5607 24933 24913 51.1 42.9 0.8 375 75.6 42.7 41 53.4 2088 3108 1809 1829 50.6 42.9 5608 2113 2094 50.1 45 0.4 305 75.4 43.3 41 52.9 2089 3109 28116 28134 50.8 47.4 5609 28505 28487 50.2 47.4 0.6 390 79.4 51.8 41 55.8 2090 3110 1808 1828 50.6 42.9 5610 2103 2082 52 45.5 1.5 296 75.5 43.6 41 53.1 2091 3111 15951 15975 53.1 40 5611 16210 16192 54.3 52.6 1.2 260 74.3 41.5 41 53.1 2092 3112 8865 8884 50.4 45 5612 9341 9322 51.1 50 0.7 477 75.6 41.7 41 53.1 2093 3113 15 33 50.7 52.6 5613 642 622 51.6 47.6 0.9 628 79 49.2 41 55.6 2094 3114 8861 8880 50.2 45 5614 9107 9086 51.6 45.5 1.4 247 73.9 40.9 41 51.9 2095 3115 1808 1828 50.6 42.9 5615 2113 2094 50.1 45 0.4 306 75.5 43.5 41 53 2096 3116 24562 24580 50.1 52.6 5616 24933 24913 51.1 42.9 1.1 372 75.5 42.5 41 53 2097 3117 28116 28134 50.8 47.4 5617 28672 28654 50.6 52.6 0.2 557 80 51.9 41 56.2 2098 3118 24560 24580 51.3 52.4 5618 25081 25063 52.4 52.6 1.1 522 75.4 41 41 53.3 2099 3119 24560 24580 51.3 52.4 5619 25079 25061 52.7 52.6 1.4 520 75.5 41.2 41 53.3 2100 3120 16366 16384 50.3 52.6 5620 16775 16755 51.1 42.9 0.7 410 75.1 41 41 52.7 2101 3121 16366 16384 50.3 52.6 5621 16774 16754 50.4 42.9 0.1 409 75.1 41.1 41 52.8 2102 3122 24569 24590 56.6 54.5 5622 25089 25070 55.8 55 0.8 521 75.4 40.9 41 54.6 2103 3123 24569 24590 56.6 54.5 5623 25088 25069 55 50 1.6 520 75.3 40.8 41 54.3 2104 3124 24567 24590 57.8 54.2 5624 25095 25072 59.3 54.2 1.5 529 75.4 40.8 41 55.2 2105 3125 24568 24591 58.9 54.2 5625 25095 25072 59.3 54.2 0.4 528 75.3 40.7 41 55.5 2106 3126 24568 24591 58.9 54.2 5626 25091 25070 59.1 54.5 0.2 524 75.4 40.8 41 55.5 2107 3127 24568 24591 58.9 54.2 5627 25090 25069 58.3 54.5 0.6 523 75.4 40.9 41 55.4 2108 3128 16366 16384 50.3 52.6 5628 16774 16753 51.1 40.9 0.8 409 75.1 41.1 41 52.8 2109 3129 1806 1825 51.1 45 5629 2103 2082 52 45.5 1 298 75.5 43.6 41 53.3 2110 3130 8374 8395 52.4 45.5 5630 9107 9086 51.6 45.5 0.8 734 75.5 40.2 41 53.4 2111 3131 24622 24643 57.1 54.5 5631 24935 24913 56.1 47.8 1 314 74.5 40.8 41 54.1 2112 3132 9130 9150 51.3 42.9 5632 9358 9338 51 42.9 0.3 229 74.5 42.8 41 52.5 2113 3133 12936 12957 53.7 45.5 5633 13530 13511 55.6 55 1.9 595 77.4 45.4 41 55.4 2114 3134 8373 8391 50.7 47.4 5634 9107 9086 51.6 45.5 0.9 735 75.4 40.1 41 53.1 2115 3135 1352 1371 56.1 55 5635 1701 1678 54.3 41.7 1.8 350 76.7 45.7 41 55.1 2116 3136 8867 8886 50.7 50 5636 9342 9323 52.1 50 1.4 476 75.7 42 41 53.3 2117 3137 1352 1371 56.1 55 5637 1701 1677 54.7 40 1.4 350 76.7 45.7 41 55.2 2118 3138 9130 9150 51.3 42.9 5638 9249 9232 50 50 1.3 120 71.7 42.5 41 50.3 2119 3139 16861 16880 50.8 50 5639 17062 17045 50.2 50 0.6 202 74.8 44.6 41 52.5 2120 3140 9130 9150 51.3 42.9 5640 9249 9231 50.8 47.4 0.5 120 71.7 42.5 41 50.5 2121 3141 9130 9150 51.3 42.9 5641 9249 9230 51.5 45 0.2 120 71.7 42.5 41 50.7 2122 3142 8372 8390 50.7 47.4 5642 9060 9039 50.3 40.9 0.4 689 75.4 40.2 41 53 2123 3143 18074 18093 50.3 45 5643 18232 18212 50.6 47.6 0.3 159 73.7 44 41 51.8 2124 3144 2671 2692 52.1 40.9 5644 3193 3172 52.6 50 0.5 523 75.8 41.9 41 53.8 2125 3145 16562 16581 52.6 50 5645 17064 17045 51.4 50 1.2 503 75.8 42.1 41 53.6 2126 3146 2671 2692 52.1 40.9 5646 3193 3173 51.4 47.6 0.7 523 75.8 41.9 41 53.6 2127 3147 8372 8390 50.7 47.4 5647 9107 9086 51.6 45.5 0.9 736 75.5 40.2 41 53.1 2128 3148 12726 12746 51.3 47.6 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41 53.7 2241 3261 13039 13058 51.8 50 5761 13749 13727 50.5 43.5 1.3 711 76.7 43.2 41 53.9 2242 3262 24096 24119 54.4 41.7 5762 24815 24792 53.4 41.7 1 720 75.8 41 41 54.2 2243 3263 17840 17859 50.8 45 5763 18229 18209 50.1 42.9 0.7 390 74.7 40.3 41 52.4 2244 3264 15255 15273 50.3 52.6 5764 15647 15628 51 45 0.7 393 75.1 41.2 41 52.7 2245 3265 988 1006 52.2 52.6 5765 1500 1482 50.6 47.4 1.6 513 76.5 43.7 41 53.8 2246 3266 24035 24053 52.2 52.6 5766 24527 24508 50.5 45 1.7 493 75.4 41.2 41 53 2247 3267 18616 18636 51.4 47.6 5767 19215 19194 50.2 40.9 1.1 600 75.7 41.2 41 53.1 2248 3268 8374 8393 51.2 45 5768 9101 9081 50.5 47.6 0.7 728 75.5 40.2 41 53.1 2249 3269 17840 17859 50.8 45 5769 18238 18219 50.3 45 0.5 399 75 40.9 41 52.7 2250 3270 24030 24047 50.7 50 5770 24526 24506 50.3 42.9 0.4 497 75.5 41.2 41 53 2251 3271 24030 24047 50.7 50 5771 24527 24507 51 42.9 0.3 498 75.4 41.2 41 53.1 2252 3272 17840 17859 50.8 45 5772 18239 18220 50 45 0.8 400 74.9 40.8 41 52.6 2253 3273 985 1008 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50 5810 24527 24508 50.5 45 0.1 690 76 41.7 41 53.4 2291 3311 23735 23752 51.2 50 5811 24013 23995 50.3 47.4 0.8 279 74.1 40.5 41 52.1 2292 3312 18080 18100 53.3 47.6 5812 18220 18202 54.8 52.6 1.5 141 73.1 44 41 52.3 2293 3313 18081 18100 51.7 50 5813 18223 18206 51.8 50 0.1 143 73.2 44.1 41 51.9 2294 3314 18081 18100 51.7 50 5814 18231 18210 52.2 45.5 0.5 151 73.6 44.4 41 52.1 2295 3315 18081 18100 51.7 50 5815 18233 18214 52 50 0.4 153 74 45.1 41 52.4 2296 3316 18081 18100 51.7 50 5816 18233 18215 51.3 52.6 0.4 153 74 45.1 41 52.3 2297 3317 8911 8928 51.9 50 5817 9252 9235 50.1 50 1.8 342 75 41.5 41 52.6 2298 3318 17791 17811 50 42.9 5818 18223 18206 51.8 50 1.7 433 74.9 40.4 41 52.5 2299 3319 8911 8928 51.9 50 5819 9249 9231 50.8 47.4 1 339 75 41.6 41 52.8 2300 3320 12352 12375 52.9 41.7 5820 12912 12892 53.6 52.4 0.8 561 76.2 42.6 41 54.3 2301 3321 8911 8928 51.9 50 5821 9249 9230 51.5 45 0.3 339 75 41.6 41 53 2302 3322 12352 12375 52.9 41.7 5822 12995 12976 51.1 45 1.8 644 76.4 42.9 41 53.9 2303 3323 17791 17811 50 42.9 5823 18233 18215 51.3 52.6 1.3 443 75.1 40.9 41 52.7 2304 3324 12977 12996 50.2 40 5824 13328 13307 51.2 45.5 1 352 76 44 41 53.4 2305 3325 12977 12996 50.2 40 5825 13329 13308 50.5 40.9 0.3 353 76 43.9 41 53.4 2306 3326 8911 8928 51.9 50 5826 9245 9226 50 45 1.8 335 74.8 41.2 41 52.5 2307 3327 8913 8931 55.5 52.6 5827 9252 9231 54 45.5 1.5 340 74.9 41.5 41 53.7 2308 3328 1402 1425 52.8 41.7 5828 1501 1480 51.9 40.9 0.9 100 72 46 41 51.1 2309 3329 24941 24960 52 50 5829 25646 25627 50.5 45 1.5 706 75.4 40.2 41 53 2310 3330 17608 17628 50.9 42.9 5830 17769 17749 50 42.9 0.9 162 72.4 40.7 41 50.8 2311 3331 24941 24960 52 50 5831 25404 25386 52.7 52.6 0.7 464 75.3 41.2 41 53.4 2312 3332 24941 24960 52 50 5832 25401 25383 50.6 47.4 1.4 461 75.2 41 41 53 2313 3333 24941 24960 52 50 5833 25400 25382 51.4 52.6 0.6 460 75.3 41.1 41 53.2 2314 3334 17608 17628 50.9 42.9 5834 18231 18210 52.2 45.5 1.2 624 75.2 40.1 41 53.1 2315 3335 18081 18099 51.2 52.6 5835 18232 18212 50.6 47.6 0.6 152 73.8 44.7 41 51.9 2316 3336 7725 7742 50 50 5836 7853 7833 50.7 47.6 0.7 129 71.2 40.3 41 49.9 2317 3337 8913 8931 55.5 52.6 5837 9252 9230 54.5 43.5 1 340 74.9 41.5 41 53.9 2318 3338 17608 17628 50.9 42.9 5838 18233 18215 51.3 52.6 0.4 626 75.3 40.3 41 53.1 2319 3339 19715 19735 52.5 47.6 5839 19931 19912 50.9 55 1.6 217 74 41.9 41 52.2 2320 3340 8913 8931 55.5 52.6 5840 9248 9226 54.7 47.8 0.7 336 74.9 41.4 41 53.9 2321 3341 18081 18099 51.2 52.6 5841 18642 18622 50.5 42.9 0.7 562 76.2 42.7 41 53.6 2322 3342 2823 2844 50.4 45.5 5842 3189 3168 51 45.5 0.5 367 75.6 42.8 41 53.2 2323 3343 19715 19735 52.5 47.6 5843 19927 19908 52.1 55 0.3 213 73.9 41.8 41 52.4 2324 3344 2823 2844 50.4 45.5 5844 3190 3169 50.7 45.5 0.2 368 75.6 42.7 41 53.1 2325 3345 28936 28956 55.2 52.4 5845 29306 29287 54.6 55 0.6 371 76.6 45.3 41 55.1 2326 3346 28936 28956 55.2 52.4 5846 29306 29285 56.7 54.5 1.6 371 76.6 45.3 41 55.3 2327 3347 28523 28544 51.6 40.9 5847 29298 29280 51.4 52.6 0.2 776 78.4 47.3 41 55.4 2328 3348 24180 24199 50.3 40 5848 24933 24913 51.1 42.9 0.9 754 75.8 40.8 41 53.2 2329 3349 19715 19735 52.5 47.6 5849 19925 19905 51.4 52.4 1.1 211 73.8 41.7 41 52.2 2330 3350 4645 4665 50.2 42.9 5850 4836 4817 51.2 45 0.9 192 75 45.3 41 52.6 2331 3351 18081 18099 51.2 52.6 5851 18702 18685 50.2 50 1 622 76.2 42.4 41 53.5 2332 3352 28522 28542 50.2 42.9 5852 29298 29280 51.4 52.6 1.2 777 78.4 47.2 41 55.1 2333 3353 24179 24199 52.7 42.9 5853 24740 24717 52.5 41.7 0.2 562 76 42.2 41 54 2334 3354 19715 19735 52.5 47.6 5854 19909 19885 52.5 40 0 195 73.3 41 41 52.1 2335 3355 1810 1830 51.2 42.9 5855 2103 2082 52 45.5 0.8 294 75.5 43.5 41 53.3 2336 3356 19716 19737 52.2 45.5 5856 19909 19885 52.5 40 0.3 194 73.1 40.7 41 51.9 2337 3357 19719 19739 50.6 42.9 5857 19909 19885 52.5 40 1.9 191 72.9 40.3 41 51.3 2338 3358 12977 12996 50.2 40 5858 13332 13312 50.9 47.6 0.6 356 76.1 44.1 41 53.4 2339 3359 19721 19745 52.3 40 5859 19909 19885 52.5 40 0.2 189 73 40.7 41 51.9 2340 3360 17608 17627 50.2 45 5860 17769 17749 50 42.9 0.2 162 72.4 40.7 41 50.8 2341 3361 17608 17627 50.2 45 5861 18231 18210 52.2 45.5 2 624 75.2 40.1 41 52.8 2342 3362 19794 19813 50 50 5862 20099 20078 50.5 40.9 0.5 306 74.4 40.8 41 52.2 2343 3363 4658 4677 50.5 50 5863 5306 5289 50.8 50 0.3 649 75.5 40.7 41 53.1 2344 3364 24179 24200 53.3 40.9 5864 24580 24560 51.3 52.4 2 402 74.6 40 41 52.7 2345 3365 19794 19813 50 50 5865 19925 19906 50.1 50 0.1 132 72.8 43.9 41 51.1 2346 3366 1046 1064 51.2 47.4 5866 1498 1481 51 50 0.2 453 76.4 43.9 41 53.9 2347 3367 1046 1064 51.2 47.4 5867 1497 1480 50.3 50 0.9 452 76.5 44 41 53.7 2348 3368 2133 2152 50.7 45 5868 2675 2656 50.4 50 0.3 543 76.8 44.2 41 54 2349 3369 28965 28984 52.9 55 5869 29298 29279 52.6 55 0.3 334 76.4 45.2 41 54.4 2350 3370 24378 24397 55 55 5870 24564 24542 55 47.8 0 187 73.6 42.2 41 53.1 2351 3371 25348 25366 51.2 47.4 5871 25651 25632 52.7 50 1.6 304 74.7 41.4 41 52.7 2352 3372 19794 19813 50 50 5872 19923 19903 50.9 47.6 0.9 130 72.7 43.8 41 51 2353 3373 28967 28987 51.6 52.4 5873 29358 29339 52.8 50 1.2 392 76.5 44.6 41 54.1 2354 3374 17608 17627 50.2 45 5874 18233 18215 51.3 52.6 1.1 626 75.3 40.3 41 52.9 2355 3375 29186 29206 51.3 42.9 5875 29358 29339 52.8 50 1.5 173 74.5 45.1 41 52.6 2356 3376 24379 24398 55 55 5876 25093 25074 54.6 55 0.4 715 75.9 41.4 41 54.6 2357 3377 3170 3191 50.9 45.5 5877 3646 3625 52 40.9 1.1 477 75.7 41.9 41 53.4 2358 3378 3170 3191 50.9 45.5 5878 3647 3628 50.6 45 0.3 478 75.7 42.1 41 53.3 2359 3379 9140 9159 50.1 45 5879 9249 9230 51.5 45 1.4 110 71.3 42.7 41 50 2360 3380 12976 12995 51.1 45 5880 13326 13306 50.7 42.9 0.4 351 76.1 44.2 41 53.6 2361 3381 12976 12995 51.1 45 5881 13328 13307 51.2 45.5 0.1 353 76.1 44.2 41 53.7 2362 3382 3168 3189 51 45.5 5882 3494 3473 50.4 40.9 0.5 327 75.1 42.2 41 52.8 2363 3383 19794 19813 50 50 5883 19917 19896 50.9 45.5 0.9 124 72.3 43.5 41 50.7 2364 3384 24379 24398 55 55 5884 24517 24494 53.2 41.7 1.8 139 72.7 43.2 41 52 2365 3385 24380 24399 55 55 5885 25093 25074 54.6 55 0.4 714 76 41.5 41 54.6 2366 3386 2823 2844 50.4 45.5 5886 3201 3183 50.6 52.6 0.2 379 75.8 43 41 53.3 2367 3387 3798 3819 54.2 50 5887 4318 4294 54.4 40 0.2 521 75.4 41.1 41 54.2 2368 3388 9139 9159 52.5 47.6 5888 9852 9829 53.1 45.8 0.6 714 75.4 40.1 41 53.6 2369 3389 9139 9159 52.5 47.6 5889 9852 9828 53.6 44 1.1 714 75.4 40.1 41 53.6 2370 3390 19795 19814 50.4 45 5890 19927 19908 52.1 55 1.7 133 72.7 43.6 41 51.1 2371 3391 19795 19814 50.4 45 5891 19924 19905 50.1 50 0.3 130 72.4 43.1 41 50.8 2372 3392 12976 12995 51.1 45 5892 13329 13308 50.5 40.9 0.6 354 76.1 44.1 41 53.5 2373 3393 2133 2152 50.7 45 5893 2672 2654 50.9 52.6 0.2 540 76.8 44.3 41 54.1 2374 3394 4593 4613 51.5 47.6 5894 4836 4817 51.2 45 0.3 244 75.3 44.3 41 53.2 2375 3395 1810 1830 51.2 42.9 5895 2113 2094 50.1 45 1.1 304 75.5 43.4 41 53 2376 3396 17036 17058 53.5 47.8 5896 17483 17465 54.4 52.6 0.9 448 75.3 41.3 41 53.9 2377 3397 1046 1064 51.2 47.4 5897 1481 1463 50.5 47.4 0.6 436 76.2 43.6 41 53.6 2378 3398 9055 9079 52.8 40 5898 9255 9236 51.1 45 1.8 201 73.5 41.3 41 51.9 2379 3399 12976 12995 51.1 45 5899 13332 13312 50.9 47.6 0.2 357 76.2 44.3 41 53.7 2380 3400 8865 8884 50.4 45 5900 9311 9292 50.7 50 0.3 447 75.4 41.4 41 53 2381 3401 25363 25381 51.1 52.6 5901 25651 25632 52.7 50 1.6 289 74.3 40.8 41 52.5 2382 3402 3168 3189 51 45.5 5902 3504 3485 50.4 45 0.6 337 75.3 42.4 41 52.9 2383 3403 25363 25381 51.1 52.6 5903 25649 25629 51.5 42.9 0.3 287 74.1 40.4 41 52.3 2384 3404 29182 29202 51.2 42.9 5904 29414 29395 50.5 50 0.7 233 75.5 45.1 41 53.1 2385 3405 3031 3051 51.3 52.4 5905 3497 3478 51.3 50 0.1 467 76.3 43.5 41 53.9 2386 3406 29172 29192 51.5 42.9 5906 29412 29393 50.3 45 1.1 241 75.6 45.2 41 53.2 2387 3407 12040 12057 50.6 50 5907 12412 12392 50 42.9 0.6 373 75.9 43.4 41 53.2 2388 3408 11543 11562 50.4 40 5908 12110 12090 51.1 42.9 0.7 568 75.9 41.9 41 53.4 2389 3409 16909 16928 50.8 45 5909 17038 17021 50.7 50 0.1 130 72.7 43.8 41 51.2 2390 3410 16909 16928 50.8 45 5910 17039 17022 51.4 50 0.6 131 72.6 43.5 41 51.2 2391 3411 18077 18097 51.5 47.6 5911 18233 18214 52 50 0.5 157 73.9 44.6 41 52.3 2392 3412 18077 18097 51.5 47.6 5912 18233 18215 51.3 52.6 0.2 157 73.9 44.6 41 52.2 2393 3413 9055 9079 52.8 40 5913 9252 9234 51.4 52.6 1.4 198 73.7 41.9 41 52.1 2394 3414 25676 25697 51.9 40.9 5914 25832 25810 53.6 47.8 1.7 157 72.1 40.1 41 51.1 2395 3415 2223 2244 51.4 45.5 5915 2676 2657 50.7 50 0.7 454 76.9 45.2 41 54.2 2396 3416 619 640 50.4 45.5 5916 1171 1153 50.4 47.4 0 553 77.9 46.8 41 54.7 2397 3417 11541 11561 50.9 42.9 5917 12110 12090 51.1 42.9 0.3 570 75.9 41.9 41 53.5 2398 3418 3360 3379 50.7 45 5918 3497 3478 51.3 50 0.6 138 74 46.4 42 52.1 2399 3419 19725 19745 50 42.9 5919 19921 19901 50.2 47.6 0.1 197 73.5 41.6 42 51.6 2400 3420 19720 19740 51.3 42.9 5920 19921 19901 50.2 47.6 1.1 202 73.4 41.1 42 51.5 2401 3421 3360 3379 50.7 45 5921 3500 3481 51.2 50 0.5 141 74 46.1 42 52.1 2402 3422 19717 19738 50.8 40.9 5922 19921 19901 50.2 47.6 0.6 205 73.4 41 42 51.5 2403 3423 24562 24580 50.1 52.6 5923 25209 25190 50.6 50 0.5 648 76.1 42 42 53.4 2404 3424 24559 24579 52 52.4 5924 24740 24717 52.5 41.7 0.5 182 76 48.4 42 53.9 2405 3425 3360 3379 50.7 45 5925 3504 3485 50.4 45 0.3 145 74.2 46.2 42 52.2 2406 3426 19716 19737 52.2 45.5 5926 19921 19900 51.8 45.5 0.4 206 73.5 41.3 42 52.1 2407 3427 3232 3251 50.3 50 5927 3494 3473 50.4 40.9 0.1 263 74.3 41.4 42 52.2 2408 3428 26039 26058 54 55 5928 26657 26636 52.6 45.5 1.5 619 75.4 40.5 42 53.7 2409 3429 3232 3251 50.3 50 5929 3504 3485 50.4 45 0.1 273 74.6 41.8 42 52.4 2410 3430 19715 19735 52.5 47.6 5930 19921 19900 51.8 45.5 0.7 207 73.7 41.5 42 52.2 2411 3431 26039 26058 54 55 5931 26653 26631 53.2 43.5 0.9 615 75.3 40.3 42 53.8 2412 3432 3229 3248 50.6 50 5932 3494 3473 50.4 40.9 0.2 266 74.3 41.4 42 52.3 2413 3433 3229 3248 50.6 50 5933 3504 3485 50.4 45 0.3 276 74.5 41.7 42 52.4 2414 3434 3225 3244 52.4 55 5934 3495 3473 51.8 43.5 0.6 271 74.7 42.1 42 52.9 2415 3435 3222 3241 52 50 5935 3650 3631 53.1 50 1.1 429 75.5 42 42 53.6 2416 3436 24559 24579 52 52.4 5936 25209 25190 50.6 50 1.3 651 76.1 42.1 42 53.6 2417 3437 6158 6178 51.3 42.9 5937 6289 6267 52.2 43.5 0.9 132 71.3 40.2 42 50.4 2418 3438 19709 19730 51.3 40.9 5938 19917 19896 50.9 45.5 0.3 209 73.7 41.6 42 52 2419 3439 3223 3241 50.2 52.6 5939 3497 3478 51.3 50 1.1 275 74.7 42.2 42 52.5 2420 3440 3223 3241 50.2 52.6 5940 3646 3625 52 40.9 1.8 424 75.4 41.7 42 53 2421 3441 3223 3241 50.2 52.6 5941 3647 3628 50.6 45 0.4 425 75.5 41.9 42 53 2422 3442 3217 3237 51.8 47.6 5942 3650 3631 53.1 50 1.3 434 75.5 41.9 42 53.5 2423 3443 9352 9372 51.3 42.9 5943 10014 9996 50.7 52.6 0.6 663 75.6 40.7 42 53.2 2424 3444 23733 23752 55.6 55 5944 24022 24003 55.5 55 0.1 290 74.5 41.4 42 53.9 2425 3445 26040 26061 56.4 54.5 5945 26661 26639 55.3 47.8 1.2 622 75.5 40.7 42 54.5 2426 3446 9918 9938 51.4 47.6 5946 10608 10589 51 50 0.4 691 75.8 41.1 42 53.4 2427 3447 7724 7742 51.4 52.6 5947 7843 7825 52.8 52.6 1.3 120 70.7 40 42 50 2428 3448 26040 26061 56.4 54.5 5948 26655 26631 56.2 48 0.2 616 75.4 40.6 42 54.8 2429 3449 28117 28135 50.6 52.6 5949 28506 28488 50.2 47.4 0.4 390 79.4 51.8 42 55.8 2430 3450 3217 3236 51.1 50 5950 3497 3478 51.3 50 0.2 281 74.7 42 42 52.7 2431 3451 3217 3236 51.1 50 5951 3500 3481 51.2 50 0.1 284 74.7 41.9 42 52.7 2432 3452 3165 3187 51.6 43.5 5952 3650 3631 53.1 50 1.5 486 75.8 42.2 42 53.6 2433 3453 19709 19730 51.3 40.9 5953 19925 19906 50.1 50 1.2 217 74 41.9 42 51.9 2434 3454 9927 9945 50.8 52.6 5954 10199 10180 51.5 45 0.7 273 75.3 43.6 42 53.1 2435 3455 9929 9946 50 50 5955 10670 10649 51.3 40.9 1.3 742 75.7 40.8 42 53.1 2436 3456 19709 19730 51.3 40.9 5956 19927 19908 52.1 55 0.9 219 74 42 42 52.3 2437 3457 9934 9953 50.7 50 5957 10356 10336 52.4 47.6 1.7 423 75.6 42.1 42 53.2 2438 3458 19709 19730 51.3 40.9 5958 19930 19910 50.6 47.6 0.7 222 74 41.9 42 52.1 2439 3459 3164 3186 51.6 43.5 5959 3650 3631 53.1 50 1.5 487 75.9 42.3 42 53.7 2440 3460 3089 3110 51.8 45.5 5960 3188 3166 51.6 43.5 0.2 100 72 46 42 51 2441 3461 18979 19000 51.6 45.5 5961 19215 19194 50.2 40.9 1.4 237 73.5 40.1 42 51.6 2442 3462 26421 26441 51.5 42.9 5962 26900 26882 51.5 52.6 0.1 480 77.5 46.2 42 54.8 2443 3463 26421 26441 51.5 42.9 5963 26828 26810 52.9 52.6 1.4 408 76.6 44.9 42 54.2 2444 3464 11540 11557 50.4 50 5964 11826 11802 51.3 40 0.8 287 74.4 41.1 42 52.3 2445 3465 26421 26441 51.5 42.9 5965 26695 26678 50.5 50 1 275 74.9 42.5 42 52.7 2446 3466 11540 11557 50.4 50 5966 11819 11798 50.3 40.9 0.1 280 74.3 41.1 42 52.2 2447 3467 11540 11557 50.4 50 5967 11817 11797 50.4 42.9 0.1 278 74.3 41 42 52.2 2448 3468 23841 23859 50.5 52.6 5968 24515 24494 50.4 40.9 0.1 675 76.1 41.9 42 53.5 2449 3469 3055 3077 52.8 43.5 5969 3495 3473 51.8 43.5 0.9 441 76 43.1 42 53.9 2450 3470 3795 3813 52.1 52.6 5970 4565 4542 53.9 41.7 1.8 771 75.6 40.3 42 53.6 2451 3471 11540 11560 53.2 47.6 5971 11984 11966 53 52.6 0.2 445 75.1 40.7 42 53.6 2452 3472 11541 11561 50.9 42.9 5972 12165 12147 51.2 47.4 0.4 625 75.7 41.3 42 53.4 2453 3473 3795 3815 54.6 52.4 5973 4318 4294 54.4 40 0.2 524 75.5 41.2 42 54.3 2454 3474 7723 7741 52.2 52.6 5974 7853 7833 50.7 47.6 1.5 131 71.3 40.5 42 50.2 2455 3475 3055 3075 51.8 47.6 5975 3504 3485 50.4 45 1.4 450 76.1 43.1 42 53.5 2456 3476 3055 3074 51.1 50 5976 3494 3473 50.4 40.9 0.7 440 76 43 42 53.4 2457 3477 26421 26441 51.5 42.9 5977 26651 26631 50.2 42.9 1.3 231 73.8 41.1 42 51.8 2458 3478 28109 28130 50.2 40.9 5978 28672 28654 50.6 52.6 0.4 564 79.9 51.6 42 56.1 2459 3479 3055 3074 51.1 50 5979 3504 3485 50.4 45 0.7 450 76.1 43.1 42 53.5 2460 3480 12232 12250 51.9 52.6 5980 12412 12392 50 42.9 1.9 181 73.2 41.4 42 51.3 2461 3481 3034 3053 50.3 50 5981 3210 3190 50.5 47.6 0.2 177 74.9 45.8 42 52.6 2462 3482 3034 3053 50.3 50 5982 3494 3473 50.4 40.9 0.1 461 76.1 43.2 42 53.5 2463 3483 3034 3053 50.3 50 5983 3504 3485 50.4 45 0.1 471 76.2 43.3 42 53.5 2464 3484 12236 12256 51.2 42.9 5984 12498 12480 50 47.4 1.1 263 74.6 42.2 42 52.4 2465 3485 12352 12375 52.9 41.7 5985 12724 12705 52.4 55 0.5 373 75.7 42.9 42 53.8 2466 3486 26421 26441 51.5 42.9 5986 26585 26567 51 47.4 0.5 165 72.2 40 42 50.9 2467 3487 3031 3051 51.3 52.4 5987 3503 3484 51.5 50 0.1 473 76.3 43.6 42 53.9 2468 3488 18704 18724 50.8 47.6 5988 19480 19459 50.3 40.9 0.4 777 75.5 40.2 42 53.1 2469 3489 3016 3036 50.2 42.9 5989 3646 3625 52 40.9 1.8 631 76.4 42.8 42 53.6 2470 3490 2823 2844 50.4 45.5 5990 3053 3034 50.3 50 0.2 231 74 41.6 42 52 2471 3491 12366 12384 51.7 52.6 5991 12994 12976 50.3 47.4 1.3 629 76.4 42.9 42 53.7 2472 3492 12366 12384 51.7 52.6 5992 12992 12974 51.2 52.6 0.5 627 76.5 43.1 42 54 2473 3493 2823 2844 50.4 45.5 5993 3056 3037 52.1 55 1.6 234 74.2 41.9 42 52.2 2474 3494 2522 2541 51.4 45 5994 2672 2654 50.9 52.6 0.5 151 75.3 48.3 42 53 2475 3495 2522 2541 51.4 45 5995 2675 2656 50.4 50 1 154 75.2 48.1 42 52.9 2476 3496 2429 2447 50.2 47.4 5996 3056 3037 52.1 55 1.9 628 76.3 42.7 42 53.6 2477 3497 2429 2447 50.2 47.4 5997 3190 3169 50.7 45.5 0.5 762 76.6 42.9 42 53.8 2478 3498 27436 27455 52.7 45 5998 27541 27521 51.7 47.6 1 106 72.1 45.3 42 51.1 2479 3499 2429 2447 50.2 47.4 5999 3192 3171 51.9 50 1.7 764 76.7 43.1 42 53.8 2480 3500 2427 2445 52.1 52.6 6000 3056 3037 52.1 55 0 630 76.4 42.9 42 54.2 2481 3501 27389 27407 50.6 47.4 6001 27541 27521 51.7 47.6 1.1 153 73.2 43.1 42 51.5 2482 3502 2427 2445 52.1 52.6 6002 3190 3169 50.7 45.5 1.4 764 76.7 43.1 42 54 2483 3503 18616 18636 51.4 47.6 6003 19316 19295 50 40.9 1.4 701 75.4 40.2 42 52.9 2484 3504 2377 2395 52.4 52.6 6004 2672 2653 51.6 50 0.8 296 77 47.3 42 54.5 2485 3505 18591 18611 51.7 42.9 6005 19216 19195 50.2 40.9 1.4 626 75.7 41.1 42 53.1 2486 3506 12366 12384 51.7 52.6 6006 12739 12718 51 40.9 0.7 374 75.6 42.8 42 53.3 2487 3507 2377 2395 52.4 52.6 6007 2672 2654 50.9 52.6 1.5 296 77 47.3 42 54.3 2488 3508 16982 17001 51.2 55 6008 17111 17090 51.1 40.9 0.1 130 74.6 48.5 42 52.6 2489 3509 2377 2395 52.4 52.6 6009 2675 2656 50.4 50 2 299 77 47.2 42 54.1 2490 3510 18590 18608 50.6 42.1 6010 19216 19195 50.2 40.9 0.3 627 75.6 41 42 53.1 2491 3511 2377 2395 52.4 52.6 6011 2891 2873 50.8 47.4 1.6 515 76.8 44.5 42 54.1 2492 3512 8220 8240 54 47.6 6012 8935 8917 54.5 52.6 0.4 716 75.4 40.1 42 54.1 2493 3513 12370 12388 50.1 47.4 6013 12998 12979 50.1 45 0.1 629 76.4 42.9 42 53.6 2494 3514 2223 2244 51.4 45.5 6014 2675 2656 50.4 50 1 453 77 45.3 42 54.1 2495 3515 2220 2239 51.3 45 6015 2672 2654 50.9 52.6 0.4 453 77 45.3 42 54.2 2496 3516 24418 24439 52.9 45.5 6016 24936 24919 51.8 50 1.1 519 76 42.4 42 53.8 2497 3517 18586 18603 50.4 44.4 6017 19216 19195 50.2 40.9 0.2 631 75.6 40.9 42 53.1 2498 3518 2220 2239 51.3 45 6018 2675 2656 50.4 50 0.8 456 76.9 45.2 42 54.1 2499 3519 1402 1422 50.2 42.9 6019 2153 2134 50.4 45 0.2 752 76.7 43.1 42 53.8 2500 3520 1356 1375 53.8 55 6020 2153 2133 52.1 42.9 1.7 798 76.9 43.5 42 54.5

TABLE 5 Primers T_(M) Product Forward primer SEQ Reverse primer SEQ (FOR & REV) length ID NO & Co-ordinates ID NO & Co-ordinates (° C.) (bp) 6076  1-19 6171 199-183 50.1 50.3 199 6077 149-169 6172 334-315 51.5 52.4 186 6078 292-310 6173 560-541 50.8 51.1 269 6079 598-619 6174 749-731 52.6 50.6 152 6080 721-742 6175 930-912 50.4 50.3 210 6081 888-912 6176 1077-1058 52.8 51.2 190 6082  984-1003 6177 1149-1131 51.1 51.1 166 6083 1157-1175 6178 1479-1460 50.9 51.6 323 6084 1420-1441 6179 1700-1680 51.2 50.7 281 6085 1685-1707 6180 1834-1811 53.8 53.7 150 6086 1740-1764 6181 1987-1963 53.4 52.2 248 6087 2007-2025 6182 2251-2232 50.3 50.1 245 6088 2226-2245 6183 2385-2366 50.4 50.1 160 6089 2428-2446 6184 2749-2728 50.1 50.3 322 6090 2742-2763 6185 2893-2875 50.6 51.4 152 6091 2823-2844 6186 3082-3058 50.4 52.3 260 6092 3007-3031 6187 3185-3164 51.9 51.0 179 6093 3234-3254 6188 3497-3478 51.1 51.3 264 6094 3453-3476 6189 3647-3627 51.8 52.1 195 6095 3601-3622 6190 3877-3853 52.5 53.6 277 6096 4007-4027 6191 4158-4135 51.1 51.4 152 6097 4141-4165 6192 4316-4295 51.3 50.8 176 6098 4366-4387 6193 4567-4544 54.6 55.4 202 6099 4488-4508 6194 4708-4690 50.7 50.3 221 6100 4658-4677 6195 4994-4974 50.5 51.2 337 6101 4902-4922 6196 5115-5092 50.5 51.4 214 6102 5239-5260 6197 5450-5430 50.8 50.9 212 6103 5366-5389 6198 5560-5542 50.5 51.8 195 6104 5593-5612 6199 5860-5836 50.8 51.6 268 6105 6042-6062 6200 6291-6271 50.4 51.1 250 6106 6271-6291 6201 6483-6463 51.1 50.2 213 6107 7017-7040 6202 7171-7153 52.4 52.8 155 6108 7253-7272 6203 7504-7486 50.3 50.3 252 6109 7415-7434 6204 7677-7654 54.5 53.6 263 6110 7615-7635 6205 7821-7798 51.1 52.8 207 6111 7728-7746 6206 7936-7915 51.7 50.1 209 6112 7845-7867 6207 7994-7970 52.7 53.4 150 6113 8011-8029 6208 8189-8170 51.4 50.6 179 6114 8143-8166 6209 8300-8281 52.2 50.8 158 6115 8221-8239 6210 8388-8369 51.0 51.1 168 6116 8553-8575 6211 8931-8915 51.8 50.3 379 6117 8867-8886 6212 9254-9236 50.7 50.6 388 6118 9244-9267 6213 9597-9573 51.9 53.4 354 6119 9620-9640 6214 9990-9969 51.3 51.3 371 6120 10009-10027 6215 10188-10171 50.2 50.2 180 6121 10093-10113 6216 10244-10223 52.4 50.6 152 6122 10242-10265 6217 10608-10589 51.2 51.0 367 6123 10549-10571 6218 10783-10763 53.7 55.2 235 6124 10766-10785 6219 10930-10912 52.0 51.1 165 6125 11065-11085 6220 11305-11287 50.7 50.0 241 6126 11265-11287 6221 11429-11405 54.5 53.5 165 6127 11552-11571 6222 11730-11709 52.0 50.4 179 6128 11705-11726 6223 11869-11848 50.1 50.2 165 6129 11801-11824 6224 11984-11967 51.5 50.4 184 6130 12040-12058 6225 12254-12235 52.3 51.9 215 6131 12235-12253 6226 12406-12388 50.1 50.1 172 6132 12366-12384 6227 12730-12712 51.7 52.2 365 6133 12727-12748 6228 12994-12976 50.8 50.3 268 6134 12948-12966 6229 13224-13201 50.7 51.7 277 6135 13175-13196 6230 13324-13300 54.3 55.1 150 6136 13237-13258 6231 13545-13526 52.9 52.9 309 6137 13790-13810 6232 13963-13945 50.9 50.7 174 6138 14080-14098 6233 14280-14257 51.5 51.0 201 6139 14405-14427 6234 14561-14540 50.2 50.9 157 6140 14882-14906 6235 15046-15024 50.9 51.5 165 6141 14951-14976 6236 15145-15124 53.1 52.9 195 6142 15113-15134 6237 15275-15257 51.6 50.8 163 6143 15211-15230 6238 15383-15363 50.2 50.1 173 6144 15364-15387 6239 15528-15506 54.0 52.1 165 6145 15456-15477 6240 15605-15585 52.0 53.2 150 6146 15513-15532 6241 15897-15876 51.2 50.4 385 6147 15837-15856 6242 15999-15978 52.3 50.8 163 6148 16073-16096 6243 16301-16277 51.7 52.8 229 6149 16245-16266 6244 16404-16380 50.3 52.0 160 6150 16366-16385 6245 16515-16492 52.9 53.8 150 6151 16553-16571 6246 16777-16758 53.4 51.5 225 6152 16832-16852 6247 17026-17004 51.0 51.6 195 6153 16982-17001 6248 17359-17340 51.2 50.2 378 6154 17354-17372 6249 17511-17490 51.3 50.4 158 6155 17422-17443 6250 17573-17552 50.2 51.1 152 6156 17603-17623 6251 17769-17748 50.7 51.5 167 6157 17728-17746 6252 17883-17862 50.9 51.2 156 6158 18011-18030 6253 18163-18140 52.9 51.9 153 6159 18076-18098 6254 18225-18205 54.4 55.0 150 6160 18270-18292 6255 18432-18413 51.9 51.4 163 6161 18352-18373 6256 18648-18629 51.3 50.8 297 6162 18550-18571 6257 18702-18684 50.4 51.9 153 6163 18720-18738 6258 19004-18983 50.6 51.0 285 6164 18960-18981 6259 19109-19085 54.7 54.3 150 6165 19065-19089 6260 19217-19195 52.8 51.7 153 6166 19310-19329 6261 19476-19454 50.2 52.1 167 6167 19569-19589 6262 19719-19701 50.5 51.8 151 6168 19707-19731 6263 19856-19833 55.7 55.9 150 6169 19771-19792 6264 19921-19901 50.1 50.2 151 6170 19833-19851 6265 19986-19966 50.9 50.7 154

TABLE 6 Primers T_(M) Product Forward primer SEQ Reverse primer SEQ (FOR & REV) length ID NO & Co-ordinates ID NO & Co-ordinates (° C.) (bp) 6266 20110-20132 6305 20425-20404 51.9 50.9 316 6267 20468-20492 6306 20617-20596 53.2 53.5 150 6268 20557-20578 6307 20891-20871 50.4 50.6 335 6269 20838-20856 6308 21037-21015 52.5 52.0 200 6270 21096-21116 6309 21295-21272 50.1 51.7 200 6271 22173-22194 6310 22414-22395 52.4 51.0 242 6272 22320-22342 6311 22501-22479 54.8 54.3 182 6273 22532-22552 6312 22695-22675 50.6 50.0 164 6274 22712-22736 6313 22873-22852 56.7 55.5 162 6275 22842-22861 6314 23086-23067 51.0 52.8 245 6276 23151-23170 6315 23395-23376 51.4 50.3 245 6277 23307-23326 6316 23524-23501 51.1 51.1 218 6278 23615-23635 6317 23776-23758 50.7 50.2 162 6279 23838-23857 6318 23996-23977 50.4 50.6 159 6280 24030-24051 6319 24407-24386 57.6 55.7 378 6281 24388-24407 6320 24581-24563 50.4 50.1 194 6282 24559-24579 6321 24938-24921 52.0 50.4 380 6283 24922-24941 6322 25184-25166 50.1 51.2 263 6284 25201-25220 6323 25400-25382 51.1 51.4 200 6285 25363-25381 6324 25646-25627 51.1 50.5 284 6286 25656-25681 6325 25839-25814 54.5 56.4 184 6287 25761-25782 6326 25982-25961 54.6 54.3 222 6288 26039-26058 6327 26189-26166 54.0 53.0 151 6289 26184-26205 6328 26333-26310 50.9 51.8 150 6290 26422-26442 6329 26660-26641 51.3 50.2 239 6291 26571-26589 6330 26739-26715 51.7 53.2 169 6292 26733-26752 6331 26960-26941 51.1 52.2 228 6293 26866-26885 6332 27139-27117 50.7 51.9 274 6294 27300-27321 6333 27458-27439 51.2 50.2 159 6295 27361-27380 6334 27579-27558 52.4 51.1 219 6296 27718-27740 6335 27917-27901 50.7 50.0 200 6297 28041-28059 6336 28207-28189 50.8 50.8 167 6298 28166-28189 6337 28411-28393 52.2 52.9 246 6299 28395-28414 6338 28671-28653 51.5 50.2 277 6300 28654-28672 6339 28821-28800 50.6 52.3 168 6301 28867-28885 6340 29184-29166 51.5 51.6 318 6302 29183-29204 6341 29360-29342 50.4 50.4 178 6303 29262-29279 6342 29626-29606 50.1 50.2 365 6304 29538-29559 6343 29690-29670 50.0 50.4 153

TABLE 7 Primers Name SEQ ID NO: Co-ordinates AB4f 6344 19869-19888 AB5f 6345 20238-20257 BC1f 6346 20581-20600 BC2f 6347 20950-20969 BC3f 6348 21339-21358 BC4f 6349 21708-21727 BC5f 6350 22041-22060 BC6f 6351 22410-22429 BC7f 6352 22759-22778 BC8f 6353 23131-23150 BC9f 6354 23500-23519 BC10f 6355 23841-23860 BC11f 6356 24210-24229 BC12f 6357 24560-24579 BC13f 6358 24941-24960 BC14f 6359 25310-25329 BC15f 6360 25675-25694 BC16f 6361 26044-26063 BC17f 6362 26413-26432 BC18f 6363 26763-26782 BC19f 6364 27132-27151 BC20f 6365 27491-27510 BC21f 6366 27845-27864 CB1r 6367 28011-28030 CB2r 6368 27671-27690 CB3r 6369 27301-27320 CB4r 6370 26931-26950 CB5r 6371 26575-26594 CB6r 6372 26191-26210 CB7r 6373 25841-25860 CB8r 6374 25476-25495 CB9r 6375 25126-25145 CB10r 6376 24791-24810 CB11r 6377 24422-24441 CB12r 6378 24031-24050 CB13r 6379 23673-23692 CB14r 6380 23298-23317 CB15r 6381 22928-22947 CB16r 6382 22567-22586 CB17r 6383 22196-22215 CB18r 6384 21831-21850 CB19r 6385 21431-21450 CB20r 6386 21073-21092 CB21r 6387 20715-20734 BA1r 6388 20345-20364 BA2r 6389 19969-19988 BA3r 6390 19599-19618 BA4r 6391 19228-19247 BA5r 6392 18852-18871

TABLE 8 Primers Name SEQ ID NO Co-ordinates F1 6393  1-19 F2 6394 292-310 F3 6395 721-742 F4 6396 984-1003 F5 6397 1420-1441 F6 6398 1740-1764 F7 6399 2226-2245 F8 6400 2742-2763 F9 6401 3007-3031 F10 6402 3453-3476 F11 6403 4007-4027 F12 6404 4366-4387 F13 6405 4658-4677 F14 6406 5239-5260 F15 6407 5593-5612 F16 6408 6271-6291 F17 6409 7253-7272 F18 6410 7615-7635 F19 6411 7845-7867 F20 6412 8143-8166 F21 6413 8553-8575 F22 6414 9244-9267 F23 6415 10009-10027 F24 6416 10242-10265 F25 6417 10766-10785 F26 6418 11265-11287 F27 6419 11705-11726 F28 6420 12040-12058 F29 6421 12366-12384 F30 6422 12948-12966 F31 6423 13237-13258 F32 6424 14080-14098 F33 6425 14882-14906 F34 6426 15113-15134 F35 6427 15364-15387 F36 6428 15513-15532 F37 6429 16073-16096 F38 6430 16366-16385 F39 6431 16832-16852 F40 6432 17354-17372 F41 6433 17603-17623 F42 6434 18011-18030 F43 6435 18270-18292 F44 6436 18550-18571 F45 6437 18960-18981 F46 6438 19310-19329 F47 6439 19707-19731 F48 6440 19833-19851 R1 6441 334-315 R2 6442 749-731 R3 6443 1077-1058 R4 6444 1479-1460 R5 6445 1834-1811 R6 6446 2251-2232 R7 6447 2749-2728 R8 6448 3082-3058 R9 6449 3497-3478 R10 6450 3877-3853 R11 6451 4316-4295 R12 6452 4708-4690 R13 6453 5115-5092 R14 6454 5560-5542 R15 6455 6291-6271 R16 6456 7171-7153 R17 6457 7677-7654 R18 6458 7936-7915 R19 6459 8189-8170 R20 6460 8388-8369 R21 6461 9254-9236 R22 6462 9990-9969 R23 6463 10244-10223 R24 6464 10783-10763 R25 6465 11305-11287 R26 6466 11730-11709 R27 6467 11984-11967 R28 6468 12406-12388 R29 6469 12994-12976 R30 6470 13324-13300 R31 6471 13963-13945 R32 6472 14561-14540 R33 6473 15145-15124 R34 6474 15383-15363 R35 6475 15605-15585 R36 6476 15999-15978 R37 6477 16404-16380 R38 6478 16777-16758 R39 6479 17359-17340 R40 6480 17573-17552 R41 6481 17883-17862 R42 6482 18225-18205 R43 6483 18648-18629 R44 6484 19004-18983 R45 6485 19217-19195 R46 6486 19719-19701 R47 6487 19921-19901

TABLE 9 Primers Name SEQ ID NO: 1 CB12R 6488 2 R0010 6489 3 R0011 6490 4 R0012 6491 5 BNI-ED 6492 6 BNI-EU 6493 7 SAR1S-U 6494 8 SAR1As-D 6495 9 SAR1S 6496 10 SAR1As 6497 11 IN2-U 6498 12 IN4-D 6499 13 IN-2 6500 14 IN-4 6501 15 IN-6 6502 16 IN-7 6503 17 COR1-U 6504 18 COR2-D 6505 19 COR-1 6506 20 COR-2 6507 21 HKUF-U 6508 22 HKUR-D 6509 23 HKU-F 6510 24 HKU-R 6511 25 1451-D 6512 26 1451-U 6513 27 690-D 6514 28 690-U 6515 29 690-D2 6516 30 690-U2 6517 31 EMC7-D 6518 32 EMC7-U 6519 33 EMC7-D2 6520 34 EMC7-U2 6521 35 EMC8-D 6522 36 EMC8-U 6523 37 EMC8-D2 6524 38 EMC8-U2 6525 39 EMC11-D 6526 40 EMC11-U 6527 41 ORF1B-D 6528 42 ORF1B-U 6529 43 ORFS-D 6530 44 ORES-U 6531 45 E7-717F 6532 46 E8-85R 6533 47 E8-307F 6534 48 E11-771F 6535 49 E11-96R 6536 50 CON1-F 6537 51 CON1-U 6538 52 CON2-F 6539 53 CON2-R 6540 54 CON3-F 6541 55 CON3-R 6542 56 15-F 6543 57 15-R 6544 58 15-F2 6545 59 15-R2 6546 60 13-F 6547 61 13-R 6548 62 13-F2 6549 63 13-R2 6550 64 CONTIG-F 6551 65 QT3-R 6552 66 QT3-F 6553 67 QIN-R 6554 68 QIN-F 6555 69 AB1-F 6556 70 AB2-F 6557 71 AB3-F 6558 72 AB1-R 6559

TABLE 10 Features of the predicted proteins and open reading frames of the SARS virus SARS ORF Length Cleavage (SEQ ID NO) (aa) Role site Features Cons^(d*) ORF1a P28 (9766)  179 Leader protein  179 (G/G)^(#) + P65 (9767)  639 Homologue of MHV p65  818 (G/A) + cleavage product Nsp1 2422^(##) Papain like protease, 3240 (Q/S) phosphoesterase domain + (9768) cleaves the first two Zn binding domain proteins Nsp2  306 3C-like protease, cleaves 3546 (Q/G) + (9769) proteins nsp1-nsp12 Nsp3 (9770)  290 ? 3836 (Q/S) 5 TMDs + Nsp4 (9771)  83 ? 3919 (Q/A) 1 TMD + Nsp5 (9772)  198 ? 4117 (Q/N) + Nsp6 (9773)  113 ? 4230 (Q/A) + Nsp7 (9774)  139 ? 4369 (Q/S) Putative growth factor-like motif + ORF1b Nsp9 (9775)  932 RNA polymerase 5298 (Q/A) + Nsp10 (9776)  601 Putative helicase 5899 (Q/A) Metal binding domain, + Tanner et al. (2003) J Biol Chem ATP/GTP binding domain 278: 39578-82 Nsp11 (9777)  527 ? 6426 (Q/S) + Nsp12 (9778)  346 ? 6772 (Q/A) + Nsp13 (9779)  298 ? — + Structural Spike (S) Major antigenic Leader peptide, 1 TMD, 17 N- + region (6042) determinant, contains the glycosylation sites receptor-binding domain Orf3 (6043)  274 ? 2 TMDs, 1 N-glycosylation site, 10 − O-glycosylation sites Orf4 (6044)  154 ? − Envelope (E)  76 Associated with viral 1 TMD, 2 N-glycosylation sites + (6045) envelope Matrix (M)  221 Associated with viral 3 TMDs, 1 N-glycosylation site + (6046) envelope, membrane spanning protein Orf7  63 ? 1 TMD − (6047) Orf8 (6048)  122 ? 1 TMD − Orf9 (6049)  44 ? Surface-associated − Orf10  39 ? Surface-associated − Orf11(6050)  84 ? 1 N-glycosylation site − Nucleocapsid (N)  422 Associated with viral phosphoprotein + (6052) genomic RNA Orf13  98 ? 1 O-glycosylation site − TMD: predicted transmembrane domain. Cons^(d)*: + indicates presence of corresponding protein at least in one of the other coronaviruses ^(#)Alternatively, cleaved after Gly-Gly (i.e. at G/A) to give a 180mer ^(##)This 2422mer may be further cleaved after residue 1922 (Gly-2740 of SEQ ID NO: 6039) to give a 1922mer PLpro containing the Zn-binding motif (SEQ ID NO: 7254) and a 500mer.

TABLE 11 Protein homologies between SARS and other coronaviruses group 1 group 2 group 3 Proteins 229E TGV PEDV MHV BCoV AIBV REPLICASE REGION leader protein <20     <20     <20     27   <20     <20     p28 p65 homologue <20     23   23   <20     20   <20     nsp1 25.5 25.8 25.4 29   30   25   (PLP protease) nsp2 40.4 43.8 44.6 50   48.4 41   (3CL protease) nsp3 30   27   29.4 34.2 35.5 28.5 nsp4 38.6 42.2 39.8 47.5 46.1 37.3 nsp5 48.2 42.9 43.9 46.8 47.3 38.7 nsp6 45.1 38.9 45.1 45.1 46.9 39.8 nsp7 53.8 54.5 56.1 56.2 55.4 58.3 nsp9 59.8 59.6 60   67.3 66.9 62.4 (polymerase) nsp10 60.7 62   62.3 67.2 68.6 58.9 (helicase) nsp11 52.3 53.7 52.3 57.6 57.6 52   nsp12 43.1 43   45.4 45.9 45   40.2 nsp13 56.4 54.4 55.3 63   65   53.4 STRUCTURAL REGION Spike (S) 28.8  31.6* 30.3 31.1 31    32.7* Envelope (E) 33*  27.9 20   23   26.5 23.2 Matrix 30.6 32.5 34.8 40.8 41.9 32.5 glycoprotein (M) Nucleocapsid 26.9 30.1 29.5 37.3 37.4 31.5 (N) *These three alignments were obtained only on a fragment of the whole protein. Numbers indicate percentage of aminoacid identity between SARS proteins and corresponding gene products of other coronaviruses. More conserved pairs are in bold; more variable pairs are underlined.

TABLE 12 Nucleotide and aminoacid differences between five SARS isolates FRA* TOR2* Urbani* CUHK* HKU* position° base/aminoacid base/aminoacid base/aminoacid base/aminoacid base/aminoacid ORF1a 2557 A/Thr G/Ala G/Ala G/Ala G/Ala 2601 T/Val C 7746 G/Pro T 7919 C/Ala T/Val 7930 G/Asp A/Asn 8387 G/Ser C/Thr 8416 G/Arg C/Thr 9404 T/Val C/Ala 9479 T/Val C/Ala 11448 T/Ile C C C C ORF1b 13494 GT/Val AG/Ser 16622 C/Ala T 17564 T/Asp C/Glu 17846 C/Arg T 18065 G/Lys A 18965 A/Ile T T T T 19064 A/Glu G G 19084 T/Ile C/Thr C/Thr C/Thr C/Thr spike 21721 G/Gly A/Asp 22222 T/Ile C/Thr 23220 T/Ser G/Ala 24872 T/Leu C 24933 T/Phe C/Leu C/Leu C/Leu C/Leu ORF3 25298 G/Gly A/Arg 25569 T/Met A/Lys matrix 26600 T/Val C/Ala C/Ala C/Ala 26857 T/Ser C/Pro ORF10 27827 T/Cys C/Arg nucleocapsid 28268 T/Ile C/Thr C/Thr C/Thr C/Thr *SARS coronavirus FRA (accession number AY310120) SARS coronavirus TOR2 (accession number AY274119) SARS coronavirus Urbani (accession number AY278741) SARS coronavirus CUHK-W1 (accession number AY278554) SARS coronavirus HKU-39849 (accession number AY278491) °The position is based on the FRA sequence.

TABLES 13-25: T-epitope predictions for SEQ ID NOS: 6039-6050 & 6052

Epitope predictions were performed at http://www.mpiib-berlin.mpg.de/MAPPP/binding.html using a minimum score of 0.5 and the BIMAS matrix, with a maximum of 20 results being selected. The analysis revealed 9 mer and 10 mer epitopes. TABLE 13 Epitopes for SEQ ID NO: 6039 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 1867 SEQ ID NO: 7400 8% 450 2 4139 SEQ ID NO: 7401 5.55% 312.5 3 88 SEQ ID NO: 7402 4% 225 4 4249 SEQ ID NO: 7403 3.55% 200 5 4059 SEQ ID NO: 7404 2.22% 125 6 2027 SEQ ID NO: 7405 1.6% 90 7 3413 SEQ ID NO: 7406 1.11% 62.5 8 1823 SEQ ID NO: 7407 0.88% 50 9 2798 SEQ ID NO: 7408 0.88% 50 10 220 SEQ ID NO: 7409 0.8% 45 11 3738 SEQ ID NO: 7410 0.8% 45 12 4182 SEQ ID NO: 7411 0.8% 45 13 4174 SEQ ID NO: 7412 0.66% 37.5 14 1940 SEQ ID NO: 7413 0.55% 31.25 15 38 SEQ ID NO: 7414 0.48% 27 16 1231 SEQ ID NO: 7415 0.44% 25 17 1613 SEQ ID NO: 7416 0.44% 25 18 3645 SEQ ID NO: 7417 0.44% 25 19 4192 SEQ ID NO: 7418 0.44% 25 20 378 SEQ ID NO: 7419 0.4% 22.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 1867 SEQ ID NO: 7420 8% 450 2 1495 SEQ ID NO: 7421 4% 225 3 3921 SEQ ID NO: 7422 2.4% 135 4 486 SEQ ID NO: 7423 2.22% 125 5 4139 SEQ ID NO: 7424 2.22% 125 6 62 SEQ ID NO: 7425 1.6% 90 7 1190 SEQ ID NO: 7426 1.6% 90 8 1284 SEQ ID NO: 7427 1.6% 90 9 3284 SEQ ID NO: 7428 1.6% 90 10 2921 SEQ ID NO: 7429 1.2% 67.5 11 349 SEQ ID NO: 7430 0.8% 45 12 789 SEQ ID NO: 7431 0.8% 45 13 1185 SEQ ID NO: 7432 0.8% 45 14 4184 SEQ ID NO: 7433 0.8% 45 15 1313 SEQ ID NO: 7434 0.64% 36 16 3948 SEQ ID NO: 7435 0.48% 27 17 149 SEQ ID NO: 7436 0.44% 25 18 941 SEQ ID NO: 7437 0.44% 25 19 1390 SEQ ID NO: 7438 0.44% 25 20 1613 SEQ ID NO: 7439 0.44% 25 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 1010 SEQ ID NO: 7440 1.48% 180 2 3155 SEQ ID NO: 7441 1.48% 180 3 1229 SEQ ID NO: 7442 1.23% 150 4 2405 SEQ ID NO: 7443 0.88% 108 5 2 SEQ ID NO: 7444 0.74% 90 6 2304 SEQ ID NO: 7445 0.74% 90 7 2358 SEQ ID NO: 7446 0.74% 90 8 3160 SEQ ID NO: 7447 0.74% 90 9 3771 SEQ ID NO: 7448 0.74% 90 10 4007 SEQ ID NO: 7449 0.74% 90 11 3079 SEQ ID NO: 7450 0.66% 81 12 4045 SEQ ID NO: 7451 0.66% 81 13 1081 SEQ ID NO: 7452 0.49% 60 14 3268 SEQ ID NO: 7453 0.49% 60 15 4144 SEQ ID NO: 7454 0.49% 60 16 614 SEQ ID NO: 7455 0.37% 45 17 728 SEQ ID NO: 7456 0.37% 45 18 1537 SEQ ID NO: 7457 0.37% 45 19 313 SEQ ID NO: 7458 0.32% 40 20 1744 SEQ ID NO: 7459 0.32% 40 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 62 SEQ ID NO: 7460 4.44% 540 2 2151 SEQ ID NO: 7461 2.46% 300 3 633 SEQ ID NO: 7462 2.22% 270 4 1158 SEQ ID NO: 7463 2.22% 270 5 2565 SEQ ID NO: 7464 2.22% 270 6 2298 SEQ ID NO: 7465 1.77% 216 7 3159 SEQ ID NO: 7466 1.11% 135 8 640 SEQ ID NO: 7467 0.98% 120 9 2186 SEQ ID NO: 7468 0.74% 90 10 3869 SEQ ID NO: 7469 0.74% 90 11 2308 SEQ ID NO: 7470 0.66% 81 12 786 SEQ ID NO: 7471 0.55% 67.5 13 749 SEQ ID NO: 7472 0.49% 60 14 1080 SEQ ID NO: 7473 0.49% 60 15 2358 SEQ ID NO: 7474 0.49% 60 16 3955 SEQ ID NO: 7475 0.49% 60 17 714 SEQ ID NO: 7476 0.37% 45 18 1081 SEQ ID NO: 7477 0.37% 45 19 1170 SEQ ID NO: 7478 0.37% 45 20 1228 SEQ ID NO: 7479 0.37% 45 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 3797 SEQ ID NO: 7480 37.57% 600 2 4202 SEQ ID NO: 7481 37.57% 600 3 3189 SEQ ID NO: 7482 25.05% 400 4 1864 SEQ ID NO: 7483 23.14% 369.6 5 1066 SEQ ID NO: 7484 22.54% 360 6 2143 SEQ ID NO: 7485 22.54% 360 7 2693 SEQ ID NO: 7486 22.54% 360 8 1426 SEQ ID NO: 7487 18.78% 300 9 1238 SEQ ID NO: 7488 18.03% 288 10 3768 SEQ ID NO: 7489 18.03% 288 11 797 SEQ ID NO: 7490 15.03% 240 12 1882 SEQ ID NO: 7491 15.03% 240 13 1490 SEQ ID NO: 7492 13.77% 220 14 2237 SEQ ID NO: 7493 13.77% 220 15 95 SEQ ID NO: 7494 12.52% 200 16 1821 SEQ ID NO: 7495 12.52% 200 17 2289 SEQ ID NO: 7496 12.52% 200 18 3080 SEQ ID NO: 7497 12.52% 200 19 3660 SEQ ID NO: 7498 12.52% 200 20 4354 SEQ ID NO: 7499 12.52% 200 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 2143 SEQ ID NO: 7500 37.87% 604.8 2 1159 SEQ ID NO: 7501 26.30% 420 3 1650 SEQ ID NO: 7502 26.30% 420 4 1150 SEQ ID NO: 7503 18.78% 300 5 2763 SEQ ID NO: 7504 18.78% 300 6 3165 SEQ ID NO: 7505 18.78% 300 7 3201 SEQ ID NO: 7506 15.03% 240 8 3694 SEQ ID NO: 7507 15.03% 240 9 4204 SEQ ID NO: 7508 15.03% 240 10 1692 SEQ ID NO: 7509 13.77% 220 11 797 SEQ ID NO: 7510 12.52% 200 12 1610 SEQ ID NO: 7511 12.52% 200 13 1789 SEQ ID NO: 7512 12.52% 200 14 1881 SEQ ID NO: 7513 12.52% 200 15 3090 SEQ ID NO: 7514 12.52% 200 16 3763 SEQ ID NO: 7515 12.52% 200 17 2569 SEQ ID NO: 7516 11.27% 180 18 194 SEQ ID NO: 7517 9.39% 150 19 1771 SEQ ID NO: 7518 9.39% 150 20 2488 SEQ ID NO: 7519 9.39% 150 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 2308 SEQ ID NO: 7520 0.20% 8144.13515256 2 3729 SEQ ID NO: 7521 0.10% 4047.23088 3 3574 SEQ ID NO: 7522 0.09% 3547.4996634 4 3615 SEQ ID NO: 7523 0.06% 2722.682592 5 3159 SEQ ID NO: 7524 0.05% 1999.734264 6 2339 SEQ ID NO: 7525 0.03% 1551.92907744 7 2201 SEQ ID NO: 7526 0.03% 1521.53694 8 3559 SEQ ID NO: 7527 0.02% 1174.38939504 9 3085 SEQ ID NO: 7528 0.02% 1146.296448 10 4070 SEQ ID NO: 7529 0.02% 970.4103696 11 3708 SEQ ID NO: 7530 0.02% 958.92888 12 3098 SEQ ID NO: 7531 0.02% 942.678 13 1362 SEQ ID NO: 7532 0.02% 900.6984 14 3563 SEQ ID NO: 7533 0.01% 735.86016 15 3774 SEQ ID NO: 7534 0.01% 687.655656 16 4242 SEQ ID NO: 7535 0.01% 685.78272 17 2340 SEQ ID NO: 7536 0.01% 668.37342936 18 650 SEQ ID NO: 7537 0.01% 640.1983392 19 3862 SEQ ID NO: 7538 0.01% 620.57772 20 2860 SEQ ID NO: 7539 0.01% 607.88448 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 2307 SEQ ID NO: 7540 0.40% 15915.66281448 2 2201 SEQ ID NO: 7541 0.12% 4772.09313 3 3558 SEQ ID NO: 7542 0.05% 2295.04855632 4 1772 SEQ ID NO: 7543 0.04% 1759.6656 5 3087 SEQ ID NO: 7544 0.03% 1215.76896 6 2339 SEQ ID NO: 7545 0.02% 1116.29986272 7 2308 SEQ ID NO: 7546 0.02% 970.14776112 8 3061 SEQ ID NO: 7547 0.02% 836.2525104 9 2748 SEQ ID NO: 7548 0.01% 726.706344 10 3837 SEQ ID NO: 7549 0.01% 720.8292 11 59 SEQ ID NO: 7550 0.01% 650.3112 12 2877 SEQ ID NO: 7551 0.01% 620.22996 13 4114 SEQ ID NO: 7552 0.01% 559.8936 14 805 SEQ ID NO: 7553 0.01% 484.4565072 15 1655 SEQ ID NO: 7554 0.01% 437.48208 16 611 SEQ ID NO: 7555 0.00% 319.9392 17 1961 SEQ ID NO: 7556 0.00% 305.94186 18 1223 SEQ ID NO: 7557 0.00% 289.08792 19 852 SEQ ID NO: 7558 0.00% 285.67242 20 2139 SEQ ID NO: 7559 0.00% 284.845869 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 4200 SEQ ID NO: 7560 50% 18 2 281 SEQ ID NO: 7561 25% 9 3 3236 SEQ ID NO: 7562 25% 9 4 509 SEQ ID NO: 7563 16.66% 6 5 848 SEQ ID NO: 7564 16.66% 6 6 2193 SEQ ID NO: 7565 16.66% 6 7 3542 SEQ ID NO: 7566 16.66% 6 8 541 SEQ ID NO: 7567 15% 5.4 9 1748 SEQ ID NO: 7568 12.5% 4.5 10 829 SEQ ID NO: 7569 11.11% 4 11 1149 SEQ ID NO: 7570 11.11% 4 12 2027 SEQ ID NO: 7571 11.11% 4 13 2576 SEQ ID NO: 7572 11.11% 4 14 873 SEQ ID NO: 7573 8.33% 3 15 2725 SEQ ID NO: 7574 8.33% 3 16 3541 SEQ ID NO: 7575 8.33% 3 17 1837 SEQ ID NO: 7576 7.5% 2.7 18 2475 SEQ ID NO: 7577 7.5% 2.7 19 2703 SEQ ID NO: 7578 7.5% 2.7 20 1823 SEQ ID NO: 7579 6.66% 2.4 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 3541 SEQ ID NO: 7580 50% 18 2 281 SEQ ID NO: 7581 25% 9 3 1495 SEQ ID NO: 7582 25% 9 4 2303 SEQ ID NO: 7583 25% 9 5 2616 SEQ ID NO: 7584 25% 9 6 48 SEQ ID NO: 7585 16.66% 6 7 1394 SEQ ID NO: 7586 16.66% 6 8 1499 SEQ ID NO: 7587 16.66% 6 9 1862 SEQ ID NO: 7588 16.66% 6 10 1163 SEQ ID NO: 7589 11.11% 4 11 4006 SEQ ID NO: 7590 11.11% 4 12 4344 SEQ ID NO: 7591 11.11% 4 13 633 SEQ ID NO: 7592 10% 3.6 14 119 SEQ ID NO: 7593 8.33% 3 15 1190 SEQ ID NO: 7594 8.33% 3 16 1195 SEQ ID NO: 7595 8.33% 3 17 1725 SEQ ID NO: 7596 8.33% 3 18 2728 SEQ ID NO: 7597 8.33% 3 19 2895 SEQ ID NO: 7598 8.33% 3 20 3033 SEQ ID NO: 7599 8.33% 3 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 1335 SEQ ID NO: 7600 4.44% 240 2 2580 SEQ ID NO: 7601 4.44% 240 3 1703 SEQ ID NO: 7602 3.70% 200 4 113 SEQ ID NO: 7603 2.22% 120 5 168 SEQ ID NO: 7604 2.22% 120 6 2842 SEQ ID NO: 7605 2.22% 120 7 4027 SEQ ID NO: 7606 2.22% 120 8 3680 SEQ ID NO: 7607 1.66% 90 9 2085 SEQ ID NO: 7608 1.48% 80 10 2492 SEQ ID NO: 7609 1.48% 80 11 2660 SEQ ID NO: 7610 1.48% 80 12 2906 SEQ ID NO: 7611 1.48% 80 13 3346 SEQ ID NO: 7612 1.48% 80 14 4038 SEQ ID NO: 7613 1.48% 80 15 1163 SEQ ID NO: 7614 1.11% 60 16 1457 SEQ ID NO: 7615 1.11% 60 17 2351 SEQ ID NO: 7616 1.11% 60 18 2471 SEQ ID NO: 7617 1.11% 60 19 3499 SEQ ID NO: 7618 1.11% 60 20 3635 SEQ ID NO: 7619 1.11% 60 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 1703 SEQ ID NO: 7620 3.70% 200 2 17 SEQ ID NO: 7621 2.22% 120 3 3008 SEQ ID NO: 7622 2.22% 120 4 4106 SEQ ID NO: 7623 2.22% 120 5 3450 SEQ ID NO: 7624 1.66% 90 6 113 SEQ ID NO: 7625 1.48% 80 7 195 SEQ ID NO: 7626 1.48% 80 8 307 SEQ ID NO: 7627 1.48% 80 9 780 SEQ ID NO: 7628 1.48% 80 10 1000 SEQ ID NO: 7629 1.48% 80 11 1072 SEQ ID NO: 7630 1.48% 80 12 1404 SEQ ID NO: 7631 1.48% 80 13 1980 SEQ ID NO: 7632 1.48% 80 14 2262 SEQ ID NO: 7633 1.48% 80 15 2543 SEQ ID NO: 7634 1.48% 80 16 2906 SEQ ID NO: 7635 1.48% 80 17 3077 SEQ ID NO: 7636 1.48% 80 18 3175 SEQ ID NO: 7637 1.48% 80 19 4195 SEQ ID NO: 7638 1.48% 80 20 4251 SEQ ID NO: 7639 1.48% 80

TABLE 14 Epitopes for SEQ ID NO: 6040 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 20 SEQ ID NO: 7640 0.04% 2.25 2 91 SEQ ID NO: 7641 0.01% 1 3 125 SEQ ID NO: 7642 0.01% 0.75 4 56 SEQ ID NO: 7643 0.00% 0.5 5 145 SEQ ID NO: 7644 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 20 SEQ ID NO: 7645 0.01% 0.9 2 56 SEQ ID NO: 7646 0.00% 0.5 3 71 SEQ ID NO: 7647 0.00% 0.5 4 144 SEQ ID NO: 7648 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 115 SEQ ID NO: 7649 0.24% 30 2 87 SEQ ID NO: 7650 0.04% 6 3 80 SEQ ID NO: 7651 0.03% 4.05 4 125 SEQ ID NO: 7652 0.01% 1.8 5 39 SEQ ID NO: 7653 0.01% 1.5 6 56 SEQ ID NO: 7654 0.01% 1.5 7 135 SEQ ID NO: 7655 0.00% 1.2 8 91 SEQ ID NO: 7656 0.00% 1 9 119 SEQ ID NO: 7657 0.00% 1 10 141 SEQ ID NO: 7658 0.00% 0.9 11 150 SEQ ID NO: 7659 0.00% 0.6 12 137 SEQ ID NO: 7660 0.00% 0.54 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 36 SEQ ID NO: 7661 0.24% 30 2 144 SEQ ID NO: 7662 0.06% 8 3 101 SEQ ID NO: 7663 0.03% 4 4 99 SEQ ID NO: 7664 0.02% 3.6 5 80 SEQ ID NO: 7665 0.02% 2.7 6 125 SEQ ID NO: 7666 0.01% 1.6875 7 71 SEQ ID NO: 7667 0.01% 1.5 8 118 SEQ ID NO: 7668 0.01% 1.5 9 40 SEQ ID NO: 7669 0.01% 1.35 10 5 SEQ ID NO: 7670 0.00% 0.9 11 56 SEQ ID NO: 7671 0.00% 0.9 12 107 SEQ ID NO: 7672 0.00% 0.6 13 135 SEQ ID NO: 7673 0.00% 0.6 14 141 SEQ ID NO: 7674 0.00% 0.6 15 148 SEQ ID NO: 7675 0.00% 0.6 16 116 SEQ ID NO: 7676 0.00% 0.5 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 153 SEQ ID NO: 7677 1.05% 16.8 2 80 SEQ ID NO: 7678 0.75% 12 3 123 SEQ ID NO: 7679 0.50% 8 4 137 SEQ ID NO: 7680 0.50% 8 5 9 SEQ ID NO: 7681 0.45% 7.2 6 77 SEQ ID NO: 7682 0.45% 7.2 7 112 SEQ ID NO: 7683 0.45% 7.2 8 73 SEQ ID NO: 7684 0.41% 6.6 9 32 SEQ ID NO: 7685 0.37% 6 10 110 SEQ ID NO: 7686 0.37% 6 11 140 SEQ ID NO: 7687 0.37% 6 12 143 SEQ ID NO: 7688 0.37% 6 13 18 SEQ ID NO: 7689 0.30% 4.8 14 54 SEQ ID NO: 7690 0.30% 4.8 15 108 SEQ ID NO: 7691 0.30% 4.8 16 141 SEQ ID NO: 7692 0.30% 4.8 17 92 SEQ ID NO: 7693 0.27% 4.4 18 33 SEQ ID NO: 7694 0.25% 4 19 49 SEQ ID NO: 7695 0.25% 4 20 111 SEQ ID NO: 7696 0.25% 4 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 142 SEQ ID NO: 7697 12.52% 200 2 110 SEQ ID NO: 7698 0.75% 12 3 99 SEQ ID NO: 7699 0.50% 8 4 8 SEQ ID NO: 7700 0.45% 7.2 5 140 SEQ ID NO: 7701 0.45% 7.2 6 32 SEQ ID NO: 7702 0.37% 6 7 17 SEQ ID NO: 7703 0.30% 4.8 8 53 SEQ ID NO: 7704 0.30% 4.8 9 76 SEQ ID NO: 7705 0.30% 4.8 10 107 SEQ ID NO: 7706 0.30% 4.8 11 111 SEQ ID NO: 7707 0.30% 4.8 12 72 SEQ ID NO: 7708 0.27% 4.4 13 91 SEQ ID NO: 7709 0.27% 4.4 14 31 SEQ ID NO: 7710 0.25% 4 15 127 SEQ ID NO: 7711 0.25% 4 16 139 SEQ ID NO: 7712 0.25% 4 17 80 SEQ ID NO: 7713 0.22% 3.6 18 38 SEQ ID NO: 7714 0.18% 3 19 118 SEQ ID NO: 7715 0.18% 3 20 49 SEQ ID NO: 7716 0.12% 2 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 80 SEQ ID NO: 7717 0.00% 171.96732 2 147 SEQ ID NO: 7718 0.00% 51.46848 3 143 SEQ ID NO: 7719 0.00% 11.6146182 4 56 SEQ ID NO: 7720 0.00% 11.304684 5 10 SEQ ID NO: 7721 0.00% 10.34586 6 6 SEQ ID NO: 7722 0.00% 6.56830734 7 26 SEQ ID NO: 7723 0.00% 6.07614 8 141 SEQ ID NO: 7724 0.00% 5.981472 9 148 SEQ ID NO: 7725 0.00% 5.194044 10 9 SEQ ID NO: 7726 0.00% 4.299183 11 137 SEQ ID NO: 7727 0.00% 4.299183 12 130 SEQ ID NO: 7728 0.00% 4.138344 13 84 SEQ ID NO: 7729 0.00% 3.42792 14 27 SEQ ID NO: 7730 0.00% 3.383484 15 2 SEQ ID NO: 7731 0.00% 3.381 16 62 SEQ ID NO: 7732 0.00% 3.251556 17 23 SEQ ID NO: 7733 0.00% 2.9542005 18 99 SEQ ID NO: 7734 0.00% 1.982232 19 33 SEQ ID NO: 7735 0.00% 1.86921 20 111 SEQ ID NO: 7736 0.00% 1.76402985 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 5 SEQ ID NO: 7737 0.00% 159.9696 2 25 SEQ ID NO: 7738 0.00% 69.552 3 80 SEQ ID NO: 7739 0.00% 36.5148 4 107 SEQ ID NO: 7740 0.00% 21.3624 5 148 SEQ ID NO: 7741 0.00% 17.73576 6 61 SEQ ID NO: 7742 0.00% 13.9104 7 147 SEQ ID NO: 7743 0.00% 11.304684 8 53 SEQ ID NO: 7744 0.00% 8.230458 9 17 SEQ ID NO: 7745 0.00% 7.3086111 10 110 SEQ ID NO: 7746 0.00% 6.174104475 11 9 SEQ ID NO: 7747 000% 6.0858 12 99 SEQ ID NO: 7748 0.00% 5.6823984 13 2 SEQ ID NO: 7749 0.00% 3.188283 14 41 SEQ ID NO: 7750 0.00% 12.206413 15 135 SEQ ID NO: 7751 0.00% 12.076624 16 76 SEQ ID NO: 7752 0.00% 2.005692 17 23 SEQ ID NO: 7753 0.00% 1.798209 18 40 SEQ ID NO: 7754 0.00% 1.68996456 19 39 SEQ ID NO: 7755 0.00% 1.516482 20 118 SEQ ID NO: 7756 0.00% 1.2683304 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 91 SEQ ID NO: 7757 2.77% 1 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 101 SEQ ID NO: 7758 33.33% 12 2 71 SEQ ID NO: 7759 2.77% 1 3 90 SEQ ID NO: 7760 1.66% 0.6 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 49 SEQ ID NO: 7761 2.22% 120 2 9 SEQ ID NO: 7762 1.11% 60 3 73 SEQ ID NO: 7763 0.66% 36 4 33 SEQ ID NO: 7764 0.37% 20 5 137 SEQ ID NO: 7765 0.37% 20 6 141 SEQ ID NO: 7766 0.37% 20 7 77 SEQ ID NO: 7767 0.22% 12 8 112 SEQ ID NO: 7768 0.22% 12 9 143 SEQ ID NO: 7769 0.22% 12 10 81 SEQ ID NO: 7770 0.14% 8 11 13 SEQ ID NO: 7771 0.09% 5 12 69 SEQ ID NO: 7772 0.09% 5 13 18 SEQ ID NO: 7773 0.07% 4 14 32 SEQ ID NO: 7774 0.07% 4 15 54 SEQ ID NO: 7775 0.07% 4 16 80 SEQ ID NO: 7776 0.07% 4 17 92 SEQ ID NO: 7777 0.07% 4 18 108 SEQ ID NO: 7778 0.07% 4 19 111 SEQ ID NO: 7779 0.07% 4 20 123 SEQ ID NO: 7780 0.07% 4 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 99 SEQ ID NO: 7781 0.74% 40 2 17 SEQ ID NO: 7782 0.37% 20 3 8 SEQ ID NO: 7783 0.22% 12 4 72 SEQ ID NO: 7784 0.22% 12 5 91 SEQ ID NO: 7785 0.22% 12 6 127 SEQ ID NO: 7786 0.11% 6 7 31 SEQ ID NO: 7787 0.07% 4 8 32 SEQ ID NO: 7788 0.07% 4 9 53 SEQ ID NO: 7789 0.07% 4 10 76 SEQ ID NO: 7790 0.07% 4 11 107 SEQ ID NO: 7791 0.07% 4 12 110 SEQ ID NO: 7792 0.07% 4 13 111 SEQ ID NO: 7793 0.07% 4 14 140 SEQ ID NO: 7794 0.07% 4 15 9 SEQ ID NO: 7795 0.05% 3 16 19 SEQ ID NO: 7796 0.05% 3 17 33 SEQ ID NO: 7797 0.03% 2 18 93 SEQ ID NO: 7798 0.03% 2 19 102 SEQ ID NO: 7799 0.03% 2 20 129 SEQ ID NO: 7800 0.02% 1.5

TABLE 15 Epitopes for SEQ ID NO: 6041 % of Start max. Rank position Sequence score Score HLA B7 - 10 mers Maximum possible score using 5625 this molecule type 1 1818 SEQ ID NO: 7801 1.6% 90 2 373 SEQ ID NO: 7802 1.33% 75 3 681 SEQ ID NO: 7803 1.33% 75 4 74 SEQ ID NO: 7804 0.88% 50 5 786 SEQ ID NO: 7805 0.88% 50 6 1495 SEQ ID NO: 7806 0.88% 50 7 88 SEQ ID NO: 7807 0.8% 45 8 357 SEQ ID NO: 7808 0.8% 45 9 1271 SEQ ID NO: 7809 0.8% 45 10 1799 SEQ ID NO: 7810 0.8% 45 11 1393 SEQ ID NO: 7811 0.48% 27 12 386 SEQ ID NO: 7812 0.44% 25 13 2304 SEQ ID NO: 7813 0.44% 25 14 198 SEQ ID NO: 7814 0.4% 22.5 15 840 SEQ ID NO: 7815 0.4% 22.5 16 2359 SEQ ID NO: 7816 0.4% 22.5 17 1194 SEQ ID NO: 7817 0.32% 18 18 1546 SEQ ID NO: 7818 0.32% 18 19 2200 SEQ ID NO: 7819 0.22% 12.5 20 996 SEQ ID NO: 7820 0.2% 11.25 HLA A1 - 9 mers Maximum possible score using 5625 this molecule type 1 995 SEQ ID NO: 7821 10% 562.5 2 1303 SEQ ID NO: 7822 2.22% 125 3 1582 SEQ ID NO: 7823 2% 112.5 4 1456 SEQ ID NO: 7824 1.6% 90 5 772 SEQ ID NO: 7825 1.11% 62.5 6 181 SEQ ID NO: 7826 0.88% 50 7 632 SEQ ID NO: 7827 0.88% 50 8 2281 SEQ ID NO: 7828 0.88% 50 9 1586 SEQ ID NO: 7829 0.8% 45 10 2109 SEQ ID NO: 7830 0.8% 45 11 745 SEQ ID NO: 7831 0.55% 31.25 12 1916 SEQ ID NO: 7832 0.53% 30 13 966 SEQ ID NO: 7833 0.44% 25 14 1387 SEQ ID NO: 7834 0.44% 25 15 2263 SEQ ID NO: 7835 0.44% 25 16 2457 SEQ ID NO: 7836 0.26% 15 17 1057 SEQ ID NO: 7837 0.22% 12.5 18 2562 SEQ ID NO: 7838 0.22% 12.5 19 74 SEQ ID NO: 7839 0.17% 10 20 298 SEQ ID NO: 7840 0.17% 10 HLA A1 - 10 mers Maximum possible score using 12150 this molecule type 1 536 SEQ ID NO: 7841 3.33% 405 2 986 SEQ ID NO: 7842 2.46% 300 3 805 SEQ ID NO: 7843 1.64% 200 4 2345 SEQ ID NO: 7844 1.48% 180 5 2481 SEQ ID NO: 7845 0.55% 67.5 6 204 SEQ ID NO: 7846 0.49% 60 7 895 SEQ ID NO: 7847 0.44% 54 8 1512 SEQ ID NO: 7848 0.44% 54 9 2491 SEQ ID NO: 7849 0.37% 45 10 436 SEQ ID NO: 7850 0.32% 40 11 917 SEQ ID NO: 7851 0.32% 40 12 1176 SEQ ID NO: 7852 0.32% 40 13 1517 SEQ ID NO: 7853 0.29% 36 14 466 SEQ ID NO: 7854 0.24% 30 15 1784 SEQ ID NO: 7855 0.24% 30 16 2039 SEQ ID NO: 7856 0.24% 30 17 2124 SEQ ID NO: 7857 0.24% 30 18 1049 SEQ ID NO: 7858 0.22% 27 19 2200 SEQ ID NO: 7859 0.22% 27 20 2598 SEQ ID NO: 7860 0.22% 27 HLA A3 - 9 mers Maximum possible score using 12150 this molecule type 1 392 SEQ ID NO: 7861 2.46% 300 2 2230 SEQ ID NO: 7862 1.48% 180 3 590 SEQ ID NO: 7863 1.11% 135 4 697 SEQ ID NO: 7864 1.11% 135 5 919 SEQ ID NO: 7865 0.74% 90 6 1354 SEQ ID NO: 7866 0.74% 90 7 1430 SEQ ID NO: 7867 0.74% 90 8 2534 SEQ ID NO: 7868 0.74% 90 9 202 SEQ ID NO: 7869 0.49% 60 10 488 SEQ ID NO: 7870 0.49% 60 11 922 SEQ ID NO: 7871 0.49% 60 12 1735 SEQ ID NO: 7872 0.49% 60 13 2281 SEQ ID NO: 7873 0.49% 60 14 1894 SEQ ID NO: 7874 0.44% 54 15 2552 SEQ ID NO: 7875 0.44% 54 16 555 SEQ ID NO: 7876 0.37% 45 17 1134 SEQ ID NO: 7877 0.37% 45 18 1149 SEQ ID NO: 7878 0.29% 36 19 283 SEQ ID NO: 7879 0.24% 30 20 917 SEQ ID NO: 7880 0.24% 30 HLA A3 - 10 mers Maximum possible score using 1596.672 this molecule type 1 2375 SEQ ID NO: 7881 36.07% 576 2 1751 SEQ ID NO: 7882 28.93% 462 3 195 SEQ ID NO: 7883 25.05% 400 4 2306 SEQ ID NO: 7884 21.04% 336 5 806 SEQ ID NO: 7885 20.66% 330 6 1252 SEQ ID NO: 7886 18.78% 300 7 160 SEQ ID NO: 7887 15.03% 240 8 517 SEQ ID NO: 7888 15.03% 240 9 375 SEQ ID NO: 7889 12.52% 200 10 1275 SEQ ID NO: 7890 12.52% 200 11 2175 SEQ ID NO: 7891 12.52% 200 12 2207 SEQ ID NO: 7892 12.52% 200 13 2343 SEQ ID NO: 7893 12.52% 200 14 443 SEQ ID NO: 7894 11.27% 180 15 668 SEQ ID NO: 7895 7.51% 120 16 1825 SEQ ID NO: 7896 6.88% 110 17 1690 SEQ ID NO: 7897 4.69% 75 18 159 SEQ ID NO: 7898 3.75% 60 19 2550 SEQ ID NO: 7899 3.75% 60 20 1949 SEQ ID NO: 7900 3.38% 54 HLA A24 - 9 mers Maximum possible score using 1596.672 this molecule type 1 641 SEQ ID NO: 7901 45.09% 720 2 809 SEQ ID NO: 7902 24.80% 396 3 1209 SEQ ID NO: 7903 22.54% 360 4 216 SEQ ID NO: 7904 18.03% 288 5 159 SEQ ID NO: 7905 15.03% 240 6 528 SEQ ID NO: 7906 15.03% 240 7 799 SEQ ID NO: 7907 15.03% 240 8 1436 SEQ ID NO: 7908 15.03% 240 9 2219 SEQ ID NO: 7909 15.03% 240 10 1065 SEQ ID NO: 7910 13.77% 220 11 1953 SEQ ID NO: 7911 13.15% 210 12 1966 SEQ ID NO: 7912 12.52% 200 13 2600 SEQ ID NO: 7913 12.52% 200 14 71 SEQ ID NO: 7914 9.39% 150 15 380 SEQ ID NO: 7915 9.39% 150 16 1989 SEQ ID NO: 7916 9.39% 150 17 342 SEQ ID NO: 7917 8.76% 140 18 1071 SEQ ID NO: 7918 8.76% 140 19 2570 SEQ ID NO: 7919 6.88% 110 20 2550 SEQ ID NO: 7920 6.26% 100 HLA A24 - 10 mers Maximum possible score using 3925227.1 this molecule type 1 1632 SEQ ID NO: 7921 0.09% 3607.31448 2 1640 SEQ ID NO: 7922 0.04% 1748.2560912 3 1776 SEQ ID NO: 7923 0.03% 1492.58592 4 2512 SEQ ID NO: 7924 0.03% 1434.16845 5 1073 SEQ ID NO: 7925 0.03% 1338.876 6 230 SEQ ID NO: 7926 0.01% 685.78272 7 1001 SEQ ID NO: 7927 0.01% 559.8936 8 716 SEQ ID NO: 7928 0.01% 558.27486 9 2280 SEQ ID NO: 7929 0.01% 511.19781048 10 590 SEQ ID NO: 7930 0.01% 469.6692 11 664 SEQ ID NO: 7931 0.01% 442.076389524 12 1094 SEQ ID NO: 7932 0.00% 382.536 13 1735 SEQ ID NO: 7933 0.00% 382.536 14 1625 SEQ ID NO: 7934 0.00% 342.4606344 15 1974 SEQ ID NO: 7935 0.00% 336.885048 16 2382 SEQ ID NO: 7936 0.00% 319.9392 17 2417 SEQ ID NO: 7937 0.00% 319.9392 18 744 SEQ ID NO: 7938 0.00% 256.416670125 19 108 SEQ ID NO: 7939 0.00% 232.52724 20 390 SEQ ID NO: 7940 0.00% 228.0411084 HLA A 0201 - 9 mers Maximum possible score using 3925227.1 this molecule type 1 2511 SEQ ID NO: 7941 0.38% 15126.90795 2 1608 SEQ ID NO: 7942 0.05% 2049.4656 3 2572 SEQ ID NO: 7943 0.04% 1879.5921264 4 255 SEQ ID NO: 7944 0.03% 1566.6522795 5 895 SEQ ID NO: 7945 0.03% 1338.876 6 1171 SEQ ID NO: 7946 0.02% 1107.960876 7 1691 SEQ ID NO: 7947 0.01% 782.95521024 8 20 SEQ ID NO: 7948 0.01% 549.9372312 9 1632 SEQ ID NO: 7949 0.01% 479.041993296 10 2280 SEQ ID NO: 7950 0.01% 472.418344576987 11 1963 SEQ ID NO: 7951 0.00% 358.73928 12 1955 SEQ ID NO: 7952 0.00% 331.093464 13 741 SEQ ID NO: 7953 0.00% 318.652488 14 523 SEQ ID NO: 7954 0.00% 278.7876 15 1073 SEQ ID NO: 7955 0.00% 266.6988828 16 2489 SEQ ID NO: 7956 0.00% 243.432 17 777 SEQ ID NO: 7957 0.00% 218.5730664 18 1737 SEQ ID NO: 7958 0.00% 218.0785572 19 589 SEQ ID NO: 7959 0.00% 210.538251 20 229 SEQ ID NO: 7960 0.00% 205.230564 HLA A 0201 - 10 mers Maximum possible score using 36 this molecule type 1 2337 SEQ ID NO: 7961 33.33% 12 2 2156 SEQ ID NO: 7962 25% 9 3 492 SEQ ID NO: 7963 20% 7.2 4 18 SEQ ID NO: 7964 16.66% 6 5 332 SEQ ID NO: 7965 16.66% 6 6 415 SEQ ID NO: 7966 16.66% 6 7 2479 SEQ ID NO: 7967 16.66% 6 8 1495 SEQ ID NO: 7968 11.11% 4 9 2035 SEQ ID NO: 7969 11.11% 4 10 1349 SEQ ID NO: 7970 10% 3.6 11 1194 SEQ ID NO: 7971 8.33% 3 12 1648 SEQ ID NO: 7972 8.33% 3 13 96 SEQ ID NO: 7973 6.66% 2.4 14 764 SEQ ID NO: 7974 6.66% 2.4 15 986 SEQ ID NO: 7975 6.66% 2.4 16 2345 SEQ ID NO: 7976 6.66% 2.4 17 698 SEQ ID NO: 7977 5.55% 2 18 1355 SEQ ID NO: 7978 5.55% 2 19 1987 SEQ ID NO: 7979 5.55% 2 20 2085 SEQ ID NO: 7980 5.55% 2 HLA A 1101 - 9 mers Maximum possible score using 36 this molecule type 1 2083 SEQ ID NO: 7981 33.33% 12 2 2123 SEQ ID NO: 7982 25% 9 3 2147 SEQ ID NO: 7983 16.66% 6 4 331 SEQ ID NO: 7984 12.5% 4.5 5 1035 SEQ ID NO: 7985 11.11% 4 6 1064 SEQ ID NO: 7986 11.11% 4 7 2154 SEQ ID NO: 7987 11.11% 4 8 1048 SEQ ID NO: 7988 7.5% 2.7 9 202 SEQ ID NO: 7989 6.66% 2.4 10 721 SEQ ID NO: 7990 6.66% 2.4 11 2109 SEQ ID NO: 7991 6.66% 2.4 12 2230 SEQ ID NO: 7992 6.66% 2.4 13 1306 SEQ ID NO: 7993 5.55% 2 14 1622 SEQ ID NO: 7994 5.55% 2 15 1772 SEQ ID NO: 7995 5.55% 2 16 1796 SEQ ID NO: 7996 5.55% 2 17 186 SEQ ID NO: 7997 5% 1.8 18 414 SEQ ID NO: 7998 5% 1.8 19 697 SEQ ID NO: 7999 5% 1.8 20 1175 SEQ ID NO: 8000 5% 1.8 HLA A 1101 - 10 mers Maximum possible score using 5400 this molecule type 1 1447 SEQ ID NO: 8001 14.81% 800 2 642 SEQ ID NO: 8002 3.70% 200 3 34 SEQ ID NO: 8003 2.22% 120 4 186 SEQ ID NO: 8004 1.48% 80 5 244 SEQ ID NO: 8005 1.48% 80 6 459 SEQ ID NO: 8006 1.48% 80 7 1475 SEQ ID NO: 8007 1.48% 80 8 1867 SEQ ID NO: 8008 1.48% 80 9 2032 SEQ ID NO: 8009 1.48% 80 10 2047 SEQ ID NO: 8010 1.48% 80 11 2335 SEQ ID NO: 8011 1.48% 80 12 622 SEQ ID NO: 8012 1.11% 60 13 1375 SEQ ID NO: 8013 1.11% 60 14 1617 SEQ ID NO: 8014 0.92% 50 15 1023 SEQ ID NO: 8015 0.83% 45 16 286 SEQ ID NO: 8016 0.74% 40 17 490 SEQ ID NO: 8017 0.74% 40 18 810 SEQ ID NO: 8018 0.74% 40 19 1420 SEQ ID NO: 8019 0.74% 40 20 1854 SEQ ID NO: 8020 0.74% 40 HLA B7 - 9 mers Maximum possible score using 5400 this molecule type 1 1617 SEQ ID NO: 8021 3.70% 200 2 752 SEQ ID NO: 8022 2.22% 120 3 1552 SEQ ID NO: 8023 2.22% 120 4 154 SEQ ID NO: 8024 1.48% 80 5 165 SEQ ID NO: 8025 1.48% 80 6 383 SEQ ID NO: 8026 1.48% 80 7 1501 SEQ ID NO: 8027 1.48% 80 8 2093 SEQ ID NO: 8028 1.48% 80 9 2564 SEQ ID NO: 8029 1.48% 80 10 622 SEQ ID NO: 8030 1.11% 60 11 1086 SEQ ID NO: 8031 1.11% 60 12 1262 SEQ ID NO: 8032 1.11% 60 13 1556 SEQ ID NO: 8033 1.11% 60 14 845 SEQ ID NO: 8034 1% 54 15 286 SEQ ID NO: 8035 0.74% 40 16 490 SEQ ID NO: 8036 0.74% 40 17 552 SEQ ID NO: 8037 0.74% 40 18 1858 SEQ ID NO: 8038 0.74% 40 19 2107 SEQ ID NO: 8039 0.74% 40 20 2582 SEQ ID NO: 8040 0.74% 40

TABLE 16 Epitopes for SEQ ID NO: 6042 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 846 SEQ ID NO: 8041 2.22% 125 2 798 SEQ ID NO: 8042 1.6% 90 3 787 SEQ ID NO: 8043 0.88% 50 4 1178 SEQ ID NO: 8044 0.88% 50 5 637 SEQ ID NO: 8045 0.8% 45 6 557 SEQ ID NO: 8046 0.44% 25 7 1020 SEQ ID NO: 8047 0.44% 25 8 282 SEQ ID NO: 8048 0.32% 18 9 1241 SEQ ID NO: 8049 0.24% 13.5 10 466 SEQ ID NO: 8050 0.22% 12.5 11 727 SEQ ID NO: 8051 0.2% 11.25 12 706 SEQ ID NO: 8052 0.17% 10 13 324 SEQ ID NO: 8053 0.16% 9 14 752 SEQ ID NO: 8054 0.16% 9 15 54 SEQ ID NO: 8055 0.13% 7.5 16 554 SEQ ID NO: 8056 0.13% 7.5 17 590 SEQ ID NO: 8057 0.12% 6.75 18 569 SEQ ID NO: 8058 0.08% 5 19 613 SEQ ID NO: 8059 0.08% 5 20 90 SEQ ID NO: 8060 0.08% 4.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 1241 SEQ ID NO: 8061 4.8% 270 2 967 SEQ ID NO: 8062 0.8% 45 3 1010 SEQ ID NO: 8063 0.48% 27 4 426 SEQ ID NO: 8064 0.44% 25 5 809 SEQ ID NO: 8065 0.44% 25 6 1178 SEQ ID NO: 8066 0.44% 25 7 787 SEQ ID NO: 8067 0.22% 12.5 8 958 SEQ ID NO: 8068 0.22% 12.5 9 727 SEQ ID NO: 8069 0.2% 11.25 10 610 SEQ ID NO: 8070 0.17% 10 11 12 SEQ ID NO: 8071 0.13% 7.5 12 1181 SEQ ID NO: 8072 0.12% 6.75 13 373 SEQ ID NO: 8073 0.11% 6.25 14 602 SEQ ID NO: 8074 0.11% 6.25 15 20 SEQ ID NO: 8075 0.04% 2.5 16 32 SEQ ID NO: 8076 0.04% 2.5 17 53 SEQ ID NO: 8077 0.04% 2.5 18 400 SEQ ID NO: 8078 0.04% 2.5 19 557 SEQ ID NO: 8079 0.04% 2.5 20 667 SEQ ID NO: 8080 0.04% 2.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 768 SEQ ID NO: 8081 0.82% 100 2 808 SEQ ID NO: 8082 0.49% 60 3 85 SEQ ID NO: 8083 0.24% 30 4 663 SEQ ID NO: 8084 0.24% 30 5 1245 SEQ ID NO: 8085 0.14% 18 6 288 SEQ ID NO: 8086 0.09% 12 7 50 SEQ ID NO: 8087 0.08% 10 8 320 SEQ ID NO: 8088 0.07% 9 9 402 SEQ ID NO: 8089 0.07% 9 10 798 SEQ ID NO: 8090 0.07% 9 11 902 SEQ ID NO: 8091 0.06% 8.1 12 364 SEQ ID NO: 8092 0.05% 6.75 13 297 SEQ ID NO: 8093 0.04% 6 14 992 SEQ ID NO: 8094 0.04% 6 15 38 SEQ ID NO: 8095 0.03% 4.5 16 249 SEQ ID NO: 8096 0.03% 4.5 17 706 SEQ ID NO: 8097 0.03% 4.05 18 1204 SEQ ID NO: 8098 0.03% 4.05 19 1178 SEQ ID NO: 8099 0.03% 4 20 343 SEQ ID NO: 8100 0.02% 3.6 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 255 SEQ ID NO: 8101 1.48% 180 2 180 SEQ ID NO: 8102 0.55% 67.5 3 768 SEQ ID NO: 8103 0.49% 60 4 1177 SEQ ID NO: 8104 0.49% 60 5 380 SEQ ID NO: 8105 0.24% 30 6 100 SEQ ID NO: 8106 0.18% 22.5 7 786 SEQ ID NO: 8107 0.16% 20 8 1217 SEQ ID NO: 8108 0.16% 20 9 207 SEQ ID NO: 8109 0.14% 18 10 1183 SEQ ID NO: 8110 0.14% 18 11 38 SEQ ID NO: 8111 0.09% 12 12 52 SEQ ID NO: 8112 0.09% 12 13 8 SEQ ID NO: 8113 0.06% 8 14 679 SEQ ID NO: 8114 0.06% 8 15 73 SEQ ID NO: 8115 0.05% 6.75 16 1204 SEQ ID NO: 8116 0.05% 6.075 17 50 SEQ ID NO: 8117 0.04% 6 18 774 SEQ ID NO: 8118 0.04% 6 19 845 SEQ ID NO: 8119 0.04% 6 20 214 SEQ ID NO: 8120 0.04% 5.4 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 1118 SEQ ID NO: 8121 19.84% 316.8 2 51 SEQ ID NO: 8122 18.78% 300 3 161 SEQ ID NO: 8123 18.78% 300 4 434 SEQ ID NO: 8124 18.78% 300 5 365 SEQ ID NO: 8125 13.77% 220 6 736 SEQ ID NO: 8126 12.52% 200 7 620 SEQ ID NO: 8127 7.51% 120 8 1068 SEQ ID NO: 8128 7.51% 120 9 817 SEQ ID NO: 8129 3.75% 60 10 336 SEQ ID NO: 8130 3.44% 55 11 687 SEQ ID NO: 8131 3.13% 50 12 254 SEQ ID NO: 8132 2.34% 37.5 13 627 SEQ ID NO: 8133 1.87% 30 14 950 SEQ ID NO: 8134 1.75% 28 15 28 SEQ ID NO: 8135 1.56% 25 16 408 SEQ ID NO: 8136 1.56% 25 17 159 SEQ ID NO: 8137 1.31% 21 18 1166 SEQ ID NO: 8138 1.26% 20.16 19 45 SEQ ID NO: 8139 1.25% 20 20 185 SEQ ID NO: 8140 1.25% 20 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 438 SEQ ID NO: 8141 27.55% 440 2 489 SEQ ID NO: 8142 22.54% 360 3 254 SEQ ID NO: 8143 18.78% 300 4 354 SEQ ID NO: 8144 11.27% 180 5 406 SEQ ID NO: 8145 11.27% 180 6 1047 SEQ ID NO: 8146 11.27% 180 7 473 SEQ ID NO: 8147 7.51% 120 8 350 SEQ ID NO: 8148 6.26% 100 9 769 SEQ ID NO: 8149 6.26% 100 10 193 SEQ ID NO: 8150 5.63% 90 11 479 SEQ ID NO: 8151 3.13% 50 12 0 SEQ ID NO: 8152 2.70% 43.2 13 813 SEQ ID NO: 8153 1.87% 30 14 739 SEQ ID NO: 8154 1.50% 24 15 782 SEQ ID NO: 8155 1.50% 24 16 1186 SEQ ID NO: 8156 1.31% 21 17 910 SEQ ID NO: 8157 1.05% 16.8 18 128 SEQ ID NO: 8158 0.93% 15 19 183 SEQ ID NO: 8159 0.93% 15 20 1069 SEQ ID NO: 8160 0.93% 15 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 1041 SEQ ID NO: 8161 0.01% 484.2379773 2 981 SEQ ID NO: 8162 0.00% 382.536 3 957 SEQ ID NO: 8163 0.00% 342.4606344 4 896 SEQ ID NO: 8164 0.00% 232.6931712 5 1173 SEQ ID NO: 8165 0.00% 201.447432 6 733 SEQ ID NO: 8166 0.00% 171.86796 7 410 SEQ ID NO: 8167 0.00% 135.45252 8 786 SEQ ID NO: 8168 0.00% 119.463012 9 150 SEQ ID NO: 8169 0.00% 102.17550222 10 1 SEQ ID NO: 8170 0.00% 94.98737754 11 595 SEQ ID NO: 8171 0.00% 93.239424 12 1095 SEQ ID NO: 8172 0.00% 89.41779 13 1166 SEQ ID NO: 8173 0.00% 87.58584 14 845 SEQ ID NO: 8174 0.00% 79.642008 15 734 SEQ ID NO: 8175 0.00% 73.47672 16 802 SEQ ID NO: 8176 0.00% 71.872056 17 1213 SEQ ID NO: 8177 0.00% 71.872056 18 105 SEQ ID NO: 8178 0.00% 50.232 19 939 SEQ ID NO: 8179 0.00% 49.13352 20 130 SEQ ID NO: 8180 0.00% 48.732354 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 372 SEQ ID NO: 8181 0.04% 1896.33528 2 410 SEQ ID NO: 8182 0.02% 1134.00849744 3 162 SEQ ID NO: 8183 0.01% 685.3897512 4 1076 SEQ ID NO: 8184 0.01% 640.90320525 5 1196 SEQ ID NO: 8185 0.01% 623.742666372 6 353 SEQ ID NO: 8186 0.01% 446.7384576 7 50 SEQ ID NO: 8187 0.00% 375.97824 8 733 SEQ ID NO: 8188 0.00% 271.863864 9 130 SEQ ID NO: 8189 0.00% 235.6873848 10 415 SEQ ID NO: 8190 0.00% 185.679 11 297 SEQ ID NO: 8191 0.00% 177.496704 12 1 SEQ ID NO: 8192 0.00% 152.42160582 13 56 SEQ ID NO: 8193 0.00% 110.013876 14 732 SEQ ID NO: 8194 0.00% 101.0988 15 6 SEQ ID NO: 8195 0.00% 98.26704 16 261 SEQ ID NO: 8196 0.00% 91.60164 17 1040 SEQ ID NO: 8197 0.00% 76.98537 18 928 SEQ ID NO: 8198 0.00% 71.2908 19 1188 SEQ ID NO: 8199 0.00% 69.81282 20 1094 SEQ ID NO: 8200 0.00% 52.5987 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 402 SEQ ID NO: 8201 25% 9 2 902 SEQ ID NO: 8202 22.5% 8.1 3 288 SEQ ID NO: 8203 11.11% 4 4 85 SEQ ID NO: 8204 6.66% 2.4 5 706 SEQ ID NO: 8205 6.66% 2.4 6 456 SEQ ID NO: 8206 5.55% 2 7 920 SEQ ID NO: 8207 5.55% 2 8 535 SEQ ID NO: 8208 5% 1.8 9 364 SEQ ID NO: 8209 3.33% 1.2 10 438 SEQ ID NO: 8210 3.33% 1.2 11 798 SEQ ID NO: 8211 3.33% 1.2 12 808 SEQ ID NO: 8212 3.33% 1.2 13 937 SEQ ID NO: 8213 3.33% 1.2 14 956 SEQ ID NO: 8214 3.33% 1.2 15 557 SEQ ID NO: 8215 2.77% 1 16 1218 SEQ ID NO: 8216 2.77% 1 17 784 SEQ ID NO: 8217 2.5% 0.9 18 249 SEQ ID NO: 8218 2.22% 0.8 19 768 SEQ ID NO: 8219 2.22% 0.8 20 1178 SEQ ID NO: 8220 2.22% 0.8 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 38 SEQ ID NO: 8221 13.33% 4.8 2 807 SEQ ID NO: 8222 12.5% 4.5 3 100 SEQ ID NO: 8223 11.11% 4 4 380 SEQ ID NO: 8224 11.11% 4 5 767 SEQ ID NO: 8225 10% 3.6 6 533 SEQ ID NO: 8226 8.33% 3 7 967 SEQ ID NO: 8227 6.66% 2.4 8 919 SEQ ID NO: 8228 5.55% 2 9 305 SEQ ID NO: 8229 5% 1.8 10 211 SEQ ID NO: 8230 3.33% 1.2 11 511 SEQ ID NO: 8231 3.33% 1.2 12 1177 SEQ ID NO: 8232 3.33% 1.2 13 429 SEQ ID NO: 8233 2.77% 1 14 758 SEQ ID NO: 8234 2.77% 1 15 797 SEQ ID NO: 8235 2.5% 0.9 16 255 SEQ ID NO: 8236 2.22% 0.8 17 986 SEQ ID NO: 8237 2.22% 0.8 18 1157 SEQ ID NO: 8238 2.22% 0.8 19 170 SEQ ID NO: 8239 1.66% 0.6 20 893 SEQ ID NO: 8240 1.66% 0.6 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 200 SEQ ID NO: 8241 1.48% 80 2 1243 SEQ ID NO: 8242 1.48% 80 3 123 SEQ ID NO: 8243 0.74% 40 4 248 SEQ ID NO: 8244 0.66% 36 5 1036 SEQ ID NO: 8245 0.66% 36 6 494 SEQ ID NO: 8246 0.37% 20 7 495 SEQ ID NO: 8247 0.37% 20 8 523 SEQ ID NO: 8248 0.37% 20 9 842 SEQ ID NO: 8249 0.37% 20 10 932 SEQ ID NO: 8250 0.37% 20 11 274 SEQ ID NO: 8251 0.33% 18 12 588 SEQ ID NO: 8252 0.22% 12 13 656 SEQ ID NO: 8253 0.22% 12 14 657 SEQ ID NO: 8254 0.22% 12 15 767 SEQ ID NO: 8255 0.22% 12 16 911 SEQ ID NO: 8256 0.22% 12 17 939 SEQ ID NO: 8257 0.22% 12 18 1007 SEQ ID NO: 8258 0.22% 12 19 1170 SEQ ID NO: 8259 0.22% 12 20 1206 SEQ ID NO: 8260 0.22% 12 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 505 SEQ ID NO: 8261 4.44% 240 2 312 SEQ ID NO: 8262 3.70% 200 3 141 SEQ ID NO: 8263 1.11% 60 4 1006 SEQ ID NO: 8264 0.66% 36 5 411 SEQ ID NO: 8265 0.44% 24 6 122 SEQ ID NO: 8266 0.37% 20 7 134 SEQ ID NO: 8267 0.37% 20 8 184 SEQ ID NO: 8268 0.37% 20 9 367 SEQ ID NO: 8269 0.37% 20 10 402 SEQ ID NO: 8270 0.37% 20 11 494 SEQ ID NO: 8271 0.37% 20 12 560 SEQ ID NO: 8272 0.37% 20 13 626 SEQ ID NO: 8273 0.37% 20 14 931 SEQ ID NO: 8274 0.37% 20 15 956 SEQ ID NO: 8275 0.37% 20 16 1117 SEQ ID NO: 8276 0.37% 20 17 1169 SEQ ID NO: 8277 0.37% 20 18 1196 SEQ ID NO: 8278 0.37% 20 19 247 SEQ ID NO: 8279 0.22% 12 20 273 SEQ ID NO: 8280 0.22% 12

TABLE 17 Epitopes for SEQ ID NO: 6043 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 168 SEQ ID NO: 8281 0.2% 11.25 2 212 SEQ ID NO: 8282 0.08% 4.5 3 223 SEQ ID NO: 8283 0.08% 4.5 4 104 SEQ ID NO: 8284 0.04% 2.5 5 170 SEQ ID NO: 8285 0.04% 2.5 6 99 SEQ ID NO: 8286 0.04% 2.25 7 188 SEQ ID NO: 8287 0.02% 1.35 8 180 SEQ ID NO: 8288 0.02% 1.25 9 219 SEQ ID NO: 8289 0.02% 1.25 10 18 SEQ ID NO: 8290 0.01% 1 11 226 SEQ ID NO: 8291 0.01% 1 12 98 SEQ ID NO: 8292 0.01% 0.625 13 151 SEQ ID NO: 8293 0.01% 0.625 14 10 SEQ ID NO: 8294 0.01% 0.6 15 13 SEQ ID NO: 8295 0.00% 0.5 16 32 SEQ ID NO: 8296 0.00% 0.5 17 70 SEQ ID NO: 8297 0.00% 0.5 18 78 SEQ ID NO: 8298 0.00% 0.5 19 82 SEQ ID NO: 8299 0.00% 0.5 20 145 SEQ ID NO: 8300 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 99 SEQ ID NO: 8301 0.8% 45 2 223 SEQ ID NO: 8302 0.8% 45 3 188 SEQ ID NO: 8303 0.48% 27 4 206 SEQ ID NO: 8304 0.2% 11.25 5 253 SEQ ID NO: 8305 0.17% 10 6 174 SEQ ID NO: 8306 0.13% 7.5 7 97 SEQ ID NO: 8307 0.04% 2.5 8 257 SEQ ID NO: 8308 0.04% 2.5 9 179 SEQ ID NO: 8309 0.04% 2.25 10 162 SEQ ID NO: 8310 0.02% 1.25 11 196 SEQ ID NO: 8311 0.02% 1.25 12 219 SEQ ID NO: 8312 0.02% 1.25 13 18 SEQ ID NO: 8313 0.01% 1 14 246 SEQ ID NO: 8314 0.01% 1 15 38 SEQ ID NO: 8315 0.01% 0.75 16 33 SEQ ID NO: 8316 0.00% 0.5 17 69 SEQ ID NO: 8317 0.00% 0.5 18 81 SEQ ID NO: 8318 0.00% 0.5 19 104 SEQ ID NO: 8319 0.00% 0.5 20 116 SEQ ID NO: 8320 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 104 SEQ ID NO: 8321 0.98% 120 2 123 SEQ ID NO: 8322 0.74% 90 3 82 SEQ ID NO: 8323 0.44% 54 4 106 SEQ ID NO: 8324 0.11% 13.5 5 99 SEQ ID NO: 8325 0.08% 10.8 6 127 SEQ ID NO: 8326 0.08% 10 7 71 SEQ ID NO: 8327 0.07% 9 8 1 SEQ ID NO: 8328 0.06% 8.1 9 113 SEQ ID NO: 8329 0.04% 6 10 84 SEQ ID NO: 8330 0.03% 4.5 11 109 SEQ ID NO: 8331 0.03% 4.05 12 58 SEQ ID NO: 8332 0.02% 3 13 138 SEQ ID NO: 8333 0.02% 3 14 44 SEQ ID NO: 8334 0.02% 2.7 15 81 SEQ ID NO: 8335 0.02% 2.7 16 226 SEQ ID NO: 8336 0.02% 2.7 17 184 SEQ ID NO: 8337 0.01% 1.8 18 102 SEQ ID NO: 8338 0.01% 1.215 19 39 SEQ ID NO: 8339 0.00% 1.2 20 234 SEQ ID NO: 8340 0.00% 0.9 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 99 SEQ ID NO: 8341 1.33% 162 2 81 SEQ ID NO: 8342 0.44% 54 3 104 SEQ ID NO: 8343 0.24% 30 4 51 SEQ ID NO: 8344 0.16% 20 5 122 SEQ ID NO: 8345 0.11% 13.5 6 71 SEQ ID NO: 8346 0.07% 9 7 69 SEQ ID NO: 8347 0.04% 6 8 223 SEQ ID NO: 8348 0.04% 5.4 9 84 SEQ ID NO: 8349 0.03% 4.5 10 63 SEQ ID NO: 8350 0.02% 3.6 11 138 SEQ ID NO: 8351 0.02% 3 12 201 SEQ ID NO: 8352 0.01% 1.8 13 44 SEQ ID NO: 8353 0.01% 1.35 14 83 SEQ ID NO: 8354 0.01% 1.35 15 116 SEQ ID NO: 8355 0.00% 1.2 16 46 SEQ ID NO: 8356 0.00% 0.9 17 183 SEQ ID NO: 8357 0.00% 0.81 18 57 SEQ ID NO: 8358 0.00% 0.6 19 93 SEQ ID NO: 8359 0.00% 0.6 20 113 SEQ ID NO: 8360 0.00% 0.6 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 198 SEQ ID NO: 8361 13.15% 210 2 105 SEQ ID NO: 8362 9.39% 150 3 210 SEQ ID NO: 8363 4.69% 75 4 75 SEQ ID NO: 8364 3.15% 50.4 5 85 SEQ ID NO: 8365 2.63% 42 6 205 SEQ ID NO: 8366 2.10% 33.6 7 77 SEQ ID NO: 8367 1.87% 30 8 158 SEQ ID NO: 8368 0.65% 10.5 9 103 SEQ ID NO: 8369 0.56% 9 10 227 SEQ ID NO: 8370 0.55% 8.8704 11 32 SEQ ID NO: 8371 0.54% 8.64 12 74 SEQ ID NO: 8372 0.50% 8 13 131 SEQ ID NO: 8373 0.50% 8 14 54 SEQ ID NO: 8374 0.46% 7.5 15 99 SEQ ID NO: 8375 0.45% 7.2 16 44 SEQ ID NO: 8376 0.37% 6 17 62 SEQ ID NO: 8377 0.37% 6 18 87 SEQ ID NO: 8378 0.37% 6 19 89 SEQ ID NO: 8379 0.37% 6 20 154 SEQ ID NO: 8380 0.37% 6 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 105 SEQ ID NO: 8381 22.54% 360 2 204 SEQ ID NO: 8382 17.53% 280 3 209 SEQ ID NO: 8383 3.13% 50 4 75 SEQ ID NO: 8384 1.87% 30 5 85 SEQ ID NO: 8385 1.87% 30 6 77 SEQ ID NO: 8386 1.12% 18 7 74 SEQ ID NO: 8387 0.84% 13.44 8 210 SEQ ID NO: 8388 0.56% 9 9 226 SEQ ID NO: 8389 0.55% 8.8704 10 98 SEQ ID NO: 8390 0.54% 8.64 11 198 SEQ ID NO: 8391 0.46% 7.5 12 67 SEQ ID NO: 8392 0.45% 7.2 13 152 SEQ ID NO: 8393 0.43% 7 14 43 SEQ ID NO: 8394 0.37% 6 15 63 SEQ ID NO: 8395 0.37% 6 16 72 SEQ ID NO: 8396 0.37% 6 17 89 SEQ ID NO: 8397 0.37% 6 18 101 SEQ ID NO: 8398 0.37% 6 19 107 SEQ ID NO: 8399 0.37% 6 20 111 SEQ ID NO: 8400 0.37% 6 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 138 SEQ ID NO: 8401 0.21% 8532.082944 2 106 SEQ ID NO: 8402 0.10% 3977.8497792 3 44 SEQ ID NO: 8403 0.03% 1243.078056 4 71 SEQ ID NO: 8404 0.00% 348.872832 5 234 SEQ ID NO: 8405 0.00% 243.432 6 51 SEQ ID NO: 8406 0.00% 130.26096 7 109 SEQ ID NO: 8407 0.00% 91.182672 8 81 SEQ ID NO: 8408 0.00% 73.342584 9 88 SEQ ID NO: 8409 0.00% 70.386624 10 1 SEQ ID NO: 8410 0.00% 65.32728732 11 38 SEQ ID NO: 8411 0.00% 47.876409 12 76 SEQ ID NO: 8412 0.00% 36.8637882 13 46 SEQ ID NO: 8413 0.00% 30.889782 14 211 SEQ ID NO: 8414 0.00% 21.616753941 15 201 SEQ ID NO: 8415 0.00% 19.657134 16 102 SEQ ID NO: 8416 0.00% 18.4318941 17 199 SEQ ID NO: 8417 0.00% 16.496865 18 74 SEQ ID NO: 8418 0.00% 15.783256167 19 62 SEQ ID NO: 8419 0.00% 13.9968225 20 99 SEQ ID NO: 8420 0.00% 10.31851392 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 78 SEQ ID NO: 8421 0.01% 556.494246 2 138 SEQ ID NO: 8422 0.01% 395.245972224 3 84 SEQ ID NO: 8423 0.00% 201.554244 4 71 SEQ ID NO: 8424 0.00% 143.65707264 5 44 SEQ ID NO: 8425 0.00% 132.54624 6 76 SEQ ID NO: 8426 0.00% 84.78671286 7 8 SEQ ID NO: 8427 0.00% 69.552 8 211 SEQ ID NO: 8428 0.00% 52.7237901 9 113 SEQ ID NO: 8429 0.00% 47.99088 10 61 SEQ ID NO: 8430 0.00% 37.4509575 11 93 SEQ ID No: 8431 0.00% 31.24872 12 137 SEQ ID NO: 8432 0.00% 31.1384304 13 37 SEQ ID NO: 8433 0.00% 27.531 14 55 SEQ ID NO: 8434 0.00% 22.9153278 15 98 SEQ ID NO: 8435 0.00% 22.1063618985 16 108 SEQ ID NO: 8436 0.00% 21.55457052 17 63 SEQ ID NO: 8437 0.00% 21.3624 18 45 SEQ ID NO: 8438 0.00% 19.657134 19 200 SEQ ID NO: 8439 0.00% 19.657134 20 104 SEQ ID NO: 8440 0.00% 13.87622016 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 58 SEQ ID NO: 8441 5.55% 2 2 125 SEQ ID NO: 8442 1.66% 0.6 3 226 SEQ ID NO: 8443 1.66% 0.6 4 229 SEQ ID NO: 8444 1.66% 0.6 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 122 SEQ ID NO: 8445 2.22% 0.8 2 228 SEQ ID NO: 8446 2.22% 0.8 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 97 SEQ ID NO: 8447 0.66% 36 2 86 SEQ ID NO: 8448 0.37% 20 3 37 SEQ ID NO: 8449 0.33% 18 4 62 SEQ ID NO: 8450 0.33% 18 5 32 SEQ ID NO: 8451 0.22% 12 6 102 SEQ ID NO: 8452 0.22% 12 7 227 SEQ ID NO: 8453 0.22% 12 8 53 SEQ ID NO: 8454 0.11% 6 9 1 SEQ ID NO: 8455 0.07% 4 10 44 SEQ ID NO: 8456 0.07% 4 11 56 SEQ ID NO: 8457 0.07% 4 12 64 SEQ ID NO: 8458 0.07% 4 13 74 SEQ ID NO: 8459 0.07% 4 14 76 SEQ ID NO: 8460 0.07% 4 15 87 SEQ ID NO: 8461 0.07% 4 16 106 SEQ ID NO: 8462 0.07% 4 17 131 SEQ ID NO: 8463 0.07% 4 18 23 SEQ ID NO: 8464 0.03% 2 19 157 SEQ ID NO: 8465 0.03% 2 20 166 SEQ ID NO: 8466 0.03% 2 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 119 SEQ ID NO: 8467 3.33% 180 2 264 SEQ ID NO: 8468 1.48% 80 3 98 SEQ ID NO: 8469 0.66% 36 4 27 SEQ ID NO: 8470 0.37% 20 5 86 SEQ ID NO: 8471 0.37% 20 6 31 SEQ ID NO: 8472 0.22% 12 7 63 SEQ ID NO: 8473 0.22% 12 8 96 SEQ ID NO: 8474 0.22% 12 9 101 SEQ ID NO: 8475 0.22% 12 10 226 SEQ ID NO: 8476 0.22% 12 11 157 SEQ ID NO: 8477 0.14% 8 12 176 SEQ ID NO: 8478 0.14% 8 13 238 SEQ ID NO: 8479 0.14% 8 14 36 SEQ ID NO: 8480 0.11% 6 15 53 SEQ ID NO: 8481 0.11% 6 16 61 SEQ ID NO: 8482 0.11% 6 17 3 SEQ ID NO: 8483 0.07% 4 18 40 SEQ ID NO: 8484 0.07% 4 19 55 SEQ ID NO: 8485 0.07% 4 20 74 SEQ ID NO: 8486 0.07% 4

TABLE 18 Epitopes for SEQ ID NO: 6044 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 69 SEQ ID NO: 8487 0.04% 2.5 2 89 SEQ ID NO: 8488 0.02% 1.5 3 141 SEQ ID NO: 8489 0.01% 1 4 113 SEQ ID NO: 8490 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 21 SEQ ID NO: 8491 0.02% 1.5 2 88 SEQ ID NO: 8492 0.02% 1.5 3 8 SEQ ID NO: 8493 0.02% 1.25 4 31 SEQ ID NO: 8494 0.00% 0.5 5 112 SEQ ID NO: 8495 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 60 SEQ ID NO: 8496 1.23% 150 2 77 SEQ ID NO: 8497 1.11% 135 3 141 SEQ ID NO: 8498 0.49% 60 4 95 SEQ ID NO: 8499 0.32% 40 5 128 SEQ ID NO: 8500 0.08% 10 6 113 SEQ ID NO: 8501 0.04% 6 7 69 SEQ ID NO: 8502 0.01% 2 8 22 SEQ ID NO: 8503 0.01% 1.8 9 42 SEQ ID NO: 8504 0.01% 1.8 10 78 SEQ ID NO: 8505 0.00% 1.2 11 32 SEQ ID NO: 8506 0.00% 1 12 54 SEQ ID NO: 8507 0.00% 0.9 13 74 SEQ ID NO: 8508 0.00% 0.9 14 28 SEQ ID NO: 8509 0.00% 0.6 15 36 SEQ ID NO: 8510 0.00% 0.6 16 48 SEQ ID NO: 8511 0.00% 0.6 17 118 SEQ ID NO: 8512 0.00% 0.6 18 4 SEQ ID NO: 8513 0.00% 0.5 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 94 SEQ ID NO: 8514 0.49% 60 2 48 SEQ ID NO: 8515 0.16% 20 3 128 SEQ ID NO: 8516 0.16% 20 4 60 SEQ ID NO: 8517 0.12% 15 5 127 SEQ ID NO: 8518 0.12% 15 6 25 SEQ ID NO: 8519 0.04% 6 7 95 SEQ ID NO: 8520 0.04% 6 8 141 SEQ ID NO: 8521 0.04% 6 9 41 SEQ ID NO: 8522 0.04% 5.4 10 77 SEQ ID NO: 8523 0.04% 5.4 11 116 SEQ ID NO: 8524 0.04% 5.4 12 91 SEQ ID NO: 8525 0.03% 4 13 4 SEQ ID NO: 8526 0.01% 2 14 112 SEQ ID NO: 8527 0.01% 1.8 15 113 SEQ ID NO: 8528 0.01% 1.35 16 12 SEQ ID NO: 8529 0.00% 1.2 17 31 SEQ ID NO: 8530 0.00% 1 18 32 SEQ ID NO: 8531 0.00% 1 19 15 SEQ ID NO: 8532 0.00% 0.9 20 27 SEQ ID NO: 8533 0.00% 0.9 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 61 SEQ ID NO: 8534 14.46% 231 2 16 SEQ ID NO: 8535 3.13% 50 3 120 SEQ ID NO: 8536 1.87% 30 4 41 SEQ ID NO: 8537 0.60% 9.6 5 71 SEQ ID NO: 8538 0.45% 7.2 6 21 SEQ ID NO: 8539 0.37% 6 7 53 SEQ ID NO: 8540 0.37% 6 8 65 SEQ ID NO: 8541 0.37% 6 9 121 SEQ ID NO: 8542 0.37% 6 10 74 SEQ ID NO: 8543 0.36% 5.76 11 20 SEQ ID NO: 8544 0.35% 5.6 12 79 SEQ ID NO: 8545 0.35% 5.6 13 105 SEQ ID NO: 8546 0.33% 5.28 14 48 SEQ ID NO: 8547 0.30% 4.8 15 88 SEQ ID NO: 8548 0.30% 4.8 16 106 SEQ ID NO: 8549 0.30% 4.8 17 37 SEQ ID NO: 8550 0.27% 4.4 18 70 SEQ ID NO: 8551 0.27% 4.4 19 18 SEQ ID NO: 8552 0.25% 4 20 57 SEQ ID NO: 8553 0.22% 3.6 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 120 SEQ ID NO: 8554 1.87% 30 2 73 SEQ ID NO: 8555 0.54% 8.64 3 19 SEQ ID NO: 8556 0.52% 8.4 4 78 SEQ ID NO: 8557 0.52% 8.4 5 104 SEQ ID NO: 8558 0.49% 7.92 6 61 SEQ ID NO: 8559 0.46% 7.5 7 47 SEQ ID NO: 8560 0.45% 7.2 8 36 SEQ ID NO: 8561 0.41% 6.6 9 52 SEQ ID NO: 8562 0.37% 6 10 64 SEQ ID NO: 8563 0.30% 4.8 11 70 SEQ ID NO: 8564 0.30% 4.8 12 105 SEQ ID NO: 8565 0.30% 4.8 13 123 SEQ ID NO: 8566 0.30% 4.8 14 69 SEQ ID NO: 8567 0.27% 4.4 15 20 SEQ ID NO: 8568 0.25% 4 16 66 SEQ ID NO: 8569 0.25% 4 17 83 SEQ ID NO: 8570 0.25% 4 18 86 SEQ ID NO: 8571 0.25% 4 19 101 SEQ ID NO: 8572 0.25% 4 20 119 SEQ ID NO: 8573 0.25% 4 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 62 SEQ ID NO: 8574 0.00% 136.1646 2 85 SEQ ID NO: 8575 0.00% 69.6969 3 47 SEQ ID NO: 8576 0.00% 60.153786 4 121 SEQ ID NO: 8577 0.00% 52.5182736 5 74 SEQ ID NO: 8578 0.00% 49.13352 6 23 SEQ ID NO: 8579 0.00% 21.99582 7 78 SEQ ID NO: 8580 0.00% 19.42488 8 114 SEQ ID NO: 8581 0.00% 14.6900655 9 4 SEQ ID NO: 8582 0.00% 11.304684 10 79 SEQ ID NO: 8583 0.00% 8.4687081 11 122 SEQ ID NO: 8584 0.00% 6.0996 12 100 SEQ ID NO: 8585 0.00% 5.382 13 105 SEQ ID NO: 8586 0.00% 4.981593 14 25 SEQ ID NO: 8587 0.00% 4.968 15 115 SEQ ID NO: 8588 0.00% 4.966482 16 24 SEQ ID NO: 8589 0.00% 4.4815221585 17 111 SEQ ID NO: 8590 0.00% 4.128201 18 94 SEQ ID NO: 8591 0.00% 3.67632 19 34 SEQ ID NO: 8592 0.00% 3.47553 20 12 SEQ ID NO: 8593 0.00% 3.30993 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 77 SEQ ID NO: 8594 0.00% 147.97188 2 62 SEQ ID NO: 8595 0.00% 143.59176 3 113 SEQ ID NO: 8596 0.00% 106.83684 4 78 SEQ ID NO: 8597 0.00% 83.526984 5 86 SEQ ID NO: 8598 0.00% 83.526984 6 74 SEQ ID NO: 8599 0.00% 69.552 7 121 SEQ ID NO: 8600 0.00% 61.06776 8 12 SEQ ID NO: 8601 0.00% 50.232 9 44 SEQ ID NO: 8602 0.00% 26.082 10 4 SEQ ID NO: 8603 0.00% 18.3816 11 0 SEQ ID NO: 8604 0.00% 17.38386 12 72 SEQ ID NO: 8605 0.00% 17.1396 13 22 SEQ ID NO: 8606 0.00% 16.21914 14 122 SEQ ID NO: 8607 0.00% 14.02908 15 64 SEQ ID NO: 8608 0.00% 11.161854 16 46 SEQ ID NO: 8609 0.00% 10.34586 17 54 SEQ ID NO: 8610 0.00% 8.846145 18 47 SEQ ID NO: 8611 0.00% 7.575080337 19 131 SEQ ID NO: 8612 0.00% 7.452 20 114 SEQ ID NO: 8613 0.00% 6.735366 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 69 SEQ ID NO: 8614 5.55% 2 2 22 SEQ ID NO: 8615 5% 1.8 3 77 SEQ ID NO: 8616 5% 1.8 4 141 SEQ ID NO: 8617 3.33% 1.2 5 60 SEQ ID NO: 8618 2.22% 0.8 6 95 SEQ ID NO: 8619 2.22% 0.8 7 36 SEQ ID NO: 8620 1.66% 0.6 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 41 SEQ ID NO: 8621 3.33% 1.2 2 68 SEQ ID NO: 8622 33.33% 1.2 3 94 SEQ ID NO: 8623 3.33% 1.2 4 31 SEQ ID NO: 8624 2.77% 1 5 127 SEQ ID NO: 8625 2.5% 0.9 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 48 SEQ ID NO: 8626 0.74% 40 2 20 SEQ ID NO: 8627 0.37% 20 3 121 SEQ ID NO: 8628 0.33% 18 4 18 SEQ ID NO: 8629 0.07% 4 5 21 SEQ ID NO: 8630 0.07% 4 6 37 SEQ ID NO: 8631 0.07% 4 7 41 SEQ ID NO: 8632 0.07% 4 8 53 SEQ ID NO: 8633 0.07% 4 9 65 SEQ ID NO: 8634 0.07% 4 10 70 SEQ ID NO: 8635 0.07% 4 11 71 SEQ ID NO: 8636 0.07% 4 12 74 SEQ ID NO: 8637 0.07% 4 13 79 SEQ ID NO: 8638 0.07% 4 14 88 SEQ ID NO: 8639 0.07% 4 15 105 SEQ ID NO: 8640 0.07% 4 16 106 SEQ ID NO: 8641 0.07% 4 17 124 SEQ ID NO: 8642 0.07% 4 18 1 SEQ ID NO: 8643 0.03% 2 19 120 SEQ ID NO: 8644 0.03% 1.8 20 11 SEQ ID NO: 8645 0.02% 1.2 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 63 SEQ ID NO: 8646 1.48% 80 2 123 SEQ ID NO: 8647 0.74% 40 3 20 SEQ ID NO: 8648 0.37% 20 4 64 SEQ ID NO: 8649 0.22% 12 5 119 SEQ ID NO: 8650 0.11% 6 6 54 SEQ ID NO: 8651 0.09% 5 7 19 SEQ ID NO: 8652 0.07% 4 8 36 SEQ ID NO: 8653 0.07% 4 9 47 SEQ ID NO: 8654 0.07% 4 10 52 SEQ ID NO: 8655 0.07% 4 11 69 SEQ ID NO: 8656 0.07% 4 12 70 SEQ ID NO: 8657 0.07% 4 13 73 SEQ ID NO: 8658 0.07% 4 14 78 SEQ ID NO: 8659 0.07% 4 15 83 SEQ ID NO: 8660 0.07% 4 16 86 SEQ ID NO: 8661 0.07% 4 17 101 SEQ ID NO: 8662 0.07% 4 18 104 SEQ ID NO: 8663 0.07% 4 19 105 SEQ ID NO: 8664 0.07% 4 20 15 SEQ ID NO: 8665 0.03% 2

TABLE 19 Epitopes for SEQ ID NO: 6045 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 4 SEQ ID NO: 8666 0.02% 1.35 2 66 SEQ ID NO: 8667 0.02% 1.35 3 33 SEQ ID NO: 8668 0.02% 1.25 4 44 SEQ ID NO: 8669 0.01% 1 5 50 SEQ ID NO: 8670 0.01% 1 6 14 SEQ ID NO: 8671 0.01% 0.75 7 48 SEQ ID NO: 8672 0.01% 0.75 8 11 SEQ ID NO: 8673 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 4 SEQ ID NO: 8674 0.12% 6.75 2 66 SEQ ID NO: 8675 0.12% 6.75 3 10 SEQ ID NO: 8676 0.00% 0.5 4 28 SEQ ID NO: 8677 0.00% 0.5 5 32 SEQ ID NO: 8678 0.00% 0.5 6 47 SEQ ID NO: 8679 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 17 SEQ ID NO: 8680 0.24% 30 2 44 SEQ ID NO: 8681 0.07% 9 3 19 SEQ ID NO: 8682 0.06% 8.1 4 50 SEQ ID NO: 8683 0.04% 5.4 5 29 SEQ ID NO: 8684 0.03% 4 6 52 SEQ ID NO: 8685 0.02% 3.24 7 54 SEQ ID NO: 8686 0.02% 3 8 11 SEQ ID NO: 8687 0.01% 1.8 9 37 SEQ ID NO: 8688 0.01% 1.8 10 25 SEQ ID NO: 8689 0.01% 1.35 11 10 SEQ ID NO: 8690 0.00% 0.9 12 16 SEQ ID NO: 8691 0.00% 0.9 13 35 SEQ ID NO: 8692 0.00% 0.6 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 49 SEQ ID NO: 8693 0.44% 54 2 17 SEQ ID NO: 8694 0.22% 27 3 10 SEQ ID NO: 8695 0.14% 18 4 16 SEQ ID NO: 8696 0.07% 9 5 32 SEQ ID NO: 8697 0.04% 6 6 19 SEQ ID NO: 8698 0.01% 1.8 7 29 SEQ ID NO: 8699 0.00% 1.2 8 23 SEQ ID NO: 8700 0.00% 0.9 9 26 SEQ ID NO: 8701 0.00% 0.9 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 18 SEQ ID NO: 8702 1.87% 30 2 24 SEQ ID NO: 8703 0.65% 10.5 3 9 SEQ ID NO: 8704 0.52% 8.4 4 12 SEQ ID NO: 8705 0.52% 8.4 5 28 SEQ ID NO: 8706 0.52% 8.4 6 42 SEQ ID NO: 8707 0.52% 8.4 7 57 SEQ ID NO: 8708 0.52% 8.4 8 66 SEQ ID NO: 8709 0.52% 8.4 9 55 SEQ ID NO: 8710 0.51% 8.25 10 0 SEQ ID NO: 8711 0.48% 7.7 11 22 SEQ ID NO: 8712 0.45% 7.2 12 10 SEQ ID NO: 8713 0.37% 6 13 25 SEQ ID NO: 8714 0.37% 6 14 30 SEQ ID NO: 8715 0.37% 6 15 19 SEQ ID NO: 8716 0.35% 5.6 16 40 SEQ ID NO: 8717 0.31% 5 17 3 SEQ ID NO: 8718 0.30% 4.8 18 65 SEQ ID NO: 8719 0.30% 4.8 19 14 SEQ ID NO: 8720 0.27% 4.32 20 56 SEQ ID NO: 8721 0.25% 4 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 55 SEQ ID NO: 8722 18.78% 300 2 18 SEQ ID NO: 8723 2.63% 42 3 21 SEQ ID NO: 8724 2.25% 36 4 2 SEQ ID NO: 8725 1.87% 30 5 24 SEQ ID NO: 8726 1.87% 30 6 11 SEQ ID NO: 8727 0.52% 8.4 7 40 SEQ ID NO: 8728 0.52% 8.4 8 65 SEQ ID NO: 8729 0.42% 6.72 9 9 SEQ ID NO: 8730 0.37% 6 10 8 SEQ ID NO: 8731 0.35% 5.6 11 27 SEQ ID NO: 8732 0.35% 5.6 12 41 SEQ ID NO: 8733 0.35% 5.6 13 57 SEQ ID NO: 8734 0.31% 5 14 17 SEQ ID NO: 8735 0.25% 4 15 29 SEQ ID NO: 8736 0.25% 4 16 64 SEQ ID NO: 8737 0.25% 4 17 16 SEQ ID NO: 8738 0.22% 3.6 18 10 SEQ ID NO: 8739 0.18% 3 19 13 SEQ ID NO: 8740 0.18% 2.88 20 23 SEQ ID NO: 8741 0.08% 1.4 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 19 SEQ ID NO: 8742 0.03% 1310.8823136 2 15 SEQ ID NO: 8743 0.02% 1082.4143022 3 16 SEQ ID NO: 8744 0.02% 1040.33238624 4 49 SEQ ID NO: 8745 0.00% 382.536 5 25 SEQ ID NO: 8746 0.00% 342.863529264 6 56 SEQ ID NO: 8747 0.00% 63.28397376 7 12 SEQ ID NO: 8748 0.00% 40.19736105 8 10 SEQ ID NO: 8749 0.00% 21.3624 9 22 SEQ ID NO: 8750 0.00% 19.7762418 10 26 SEQ ID NO: 8751 0.00% 12.6684 11 20 SEQ ID NO: 8752 0.00% 11.544666 12 37 SEQ ID NO: 8753 0.00% 10.4328 13 32 SEQ ID NO: 8754 0.00% 8.4456 14 23 SEQ ID NO: 8755 0.00% 6.2888049 15 47 SEQ ID NO: 8756 0.00% 6.0858 16 3 SEQ ID NO: 8757 0.00% 4.582929078 17 18 SEQ ID NO: 8758 0.00% 4.4855150505 18 28 SEQ ID NO: 8759 0.00% 4.2923589 19 62 SEQ ID NO: 8760 0.00% 2.88098391 20 27 SEQ ID NO: 8761 0.00% 1.699677 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 17 SEQ ID NO: 8762 0.16% 6459.14167272 2 19 SEQ ID NO: 8763 0.01% 607.88448 3 25 SEQ ID NO: 8764 0.00% 126.83304 4 11 SEQ ID NO: 8765 0.00% 63.16728165 5 15 SEQ ID NO: 8766 0.00% 53.54651988 6 37 SEQ ID NO: 8767 0.00% 28.51632 7 14 SEQ ID NO: 8768 0.00% 21.8247414 8 29 SEQ ID NO: 8769 0.00% 21.3624 9 26 SEQ ID NO: 8770 0.00% 19.42488 10 3 SEQ ID NO: 8771 0.00% 17.2167282 11 48 SEQ ID NO: 8772 0.00% 15.7068219 12 12 SEQ ID NO: 8773 0.00% 9.8581266 13 27 SEQ ID NO: 8774 0.00% 7.3086111 14 39 SEQ ID NO: 8775 0.00% 7.10976 15 23 SEQ ID NO: 8776 0.00% 5.7419523 16 22 SEQ ID NO: 8777 0.00% 4.599126 17 45 SEQ ID NO: 8778 0.00% 2.5495155 18 31 SEQ ID NO: 8779 0.00% 2.52747 19 52 SEQ ID NO: 8780 0.00% 2.383605 20 20 SEQ ID NO: 8781 0.00% 2.332847151 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 44 SEQ ID NO: 8782 3.33% 1.2 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 3 SEQ ID NO: 8783 0.37% 20 2 12 SEQ ID NO: 8784 0.37% 20 3 22 SEQ ID NO: 8785 0.37% 20 4 56 SEQ ID NO: 8786 0.37% 20 5 30 SEQ ID NO: 8787 0.22% 12 6 9 SEQ ID NO: 8788 0.07% 4 7 10 SEQ ID NO: 8789 0.07% 4 8 19 SEQ ID NO: 8790 0.07% 4 9 25 SEQ ID NO: 8791 0.07% 4 10 28 SEQ ID NO: 8792 0.07% 4 11 42 SEQ ID NO: 8793 0.07% 4 12 65 SEQ ID NO: 8794 0.07% 4 13 35 SEQ ID NO: 8795 0.05% 3 14 66 SEQ ID NO: 8796 0.02% 1.2 15 15 SEQ ID NO: 8797 0.01% 1 16 47 SEQ ID NO: 8798 0.01% 1 17 20 SEQ ID NO: 8799 0.01% 0.6 18 23 SEQ ID NO: 8800 0.00% 0.5 19 27 SEQ ID NO: 8801 0.00% 0.5 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 27 SEQ ID NO: 8802 0.37% 20 2 8 SEQ ID NO: 8803 0.07% 4 3 9 SEQ ID NO: 8804 0.07% 4 4 11 SEQ ID NO: 8805 0.07% 4 5 17 SEQ ID NO: 8806 0.07% 4 6 29 SEQ ID NO: 8807 0.07% 4 7 41 SEQ ID NO: 8808 0.07% 4 8 52 SEQ ID NO: 8809 0.07% 4 9 64 SEQ ID NO: 8810 0.07% 4 10 65 SEQ ID NO: 8811 0.07% 4 11 3 SEQ ID NO: 8812 0.03% 2 12 23 SEQ ID NO: 8813 0.03% 2 13 21 SEQ ID NO: 8814 0.02% 1.2 14 15 SEQ ID NO: 8815 0.01% 1 15 35 SEQ ID NO: 8816 0.01% 0.6 16 39 SEQ ID NO: 8817 0.01% 0.6 17 12 SEQ ID NO: 8818 0.00% 0.5 18 22 SEQ ID NO: 8819 0.00% 0.5 19 45 SEQ ID NO: 8820 0.00% 0.5

TABLE 20 Epitopes for SEQ ID NO: 6046 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 186 SEQ ID NO: 8821 2.22% 125 2 156 SEQ ID NO: 8822 0.88% 50 3 14 SEQ ID NO: 8823 0.08% 4.5 4 0 SEQ ID NO: 8824 0.04% 2.5 5 29 SEQ ID NO: 8825 0.04% 2.5 6 85 SEQ ID NO: 8826 0.04% 2.5 7 168 SEQ ID NO: 8827 0.04% 2.5 8 133 SEQ ID NO: 8828 0.02% 1.35 9 111 SEQ ID NO: 8829 0.02% 1.125 10 61 SEQ ID NO: 8830 0.01% 1 11 7 SEQ ID NO: 8831 0.01% 0.9 12 131 SEQ ID NO: 8832 0.01% 0.9 13 211 SEQ ID NO: 8833 0.01% 0.625 14 4 SEQ ID NO: 8834 0.00% 0.5 15 43 SEQ ID NO: 8835 0.00% 0.5 16 95 SEQ ID NO: 8836 0.00% 0.5 17 136 SEQ ID NO: 8837 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 133 SEQ ID NO: 8838 0.04% 2.7 2 84 SEQ ID NO: 8839 0.04% 2.5 3 167 SEQ ID NO: 8840 0.04% 2.5 4 186 SEQ ID NO: 8841 0.04% 2.5 5 131 SEQ ID NO: 8842 0.04% 2.25 6 14 SEQ ID NO: 8843 0.03% 1.8 7 205 SEQ ID NO: 8844 0.02% 1.25 8 111 SEQ ID NO: 8845 0.02% 1.125 9 60 SEQ ID NO: 8846 0.01% 1 10 188 SEQ ID NO: 8847 0.01% 0.75 11 211 SEQ ID NO: 8848 0.01% 0.625 12 26 SEQ ID NO: 8849 0.00% 0.5 13 94 SEQ ID NO: 8850 0.00% 0.5 14 135 SEQ ID NO: 8851 0.00% 0.5 15 168 SEQ ID NO: 8852 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 43 SEQ ID NO: 8853 0.24% 30 2 90 SEQ ID NO: 8854 0.14% 18 3 148 SEQ ID NO: 8855 0.09% 12 4 4 SEQ ID NO: 8856 0.05% 6.75 5 24 SEQ ID NO: 8857 0.04% 6 6 19 SEQ ID NO: 8858 0.04% 5.4 7 136 SEQ ID NO: 8859 0.04% 5.4 8 54 SEQ ID NO: 8860 0.03% 4.5 9 32 SEQ ID NO: 8861 0.03% 4 10 14 SEQ ID NO: 8862 0.02% 3.6 11 59 SEQ ID NO: 8863 0.02% 3.6 12 88 SEQ ID NO: 8864 0.02% 3 13 87 SEQ ID NO: 8865 0.02% 2.7 14 29 SEQ ID NO: 8866 0.01% 1.8 15 48 SEQ ID NO: 8867 0.01% 1.8 16 115 SEQ ID NO: 8868 0.01% 1.8 17 186 SEQ ID NO: 8869 0.01% 1.8 18 106 SEQ ID NO: 8870 0.01% 1.5 19 53 SEQ ID NO: 8871 0.01% 1.35 20 173 SEQ ID NO: 8872 0.00% 1.2 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 24 SEQ ID NO: 8873 0.22% 27 2 54 SEQ ID NO: 8874 0.18% 22.5 3 135 SEQ ID NO: 8875 0.08% 10.8 4 51 SEQ ID NO: 8876 0.07% 9 5 13 SEQ ID NO: 8877 0.06% 8.1 6 26 SEQ ID NO: 8878 0.04% 6 7 31 SEQ ID NO: 8879 0.04% 6 8 90 SEQ ID NO: 8880 0.04% 6 9 43 SEQ ID NO: 8881 0.03% 4.5 10 19 SEQ ID NO: 8882 0.03% 4.05 11 169 SEQ ID NO: 8883 0.02% 3 12 87 SEQ ID NO: 8884 0.02% 2.7 13 84 SEQ ID NO: 8885 0.01% 1.8 14 88 SEQ ID NO: 8886 0.01% 1.8 15 94 SEQ ID NO: 8887 0.01% 1.8 16 64 SEQ ID NO: 8888 0.00% 1.2 17 131 SEQ ID NO: 8889 0.00% 1.2 18 99 SEQ ID NO: 8890 0.00% 1 19 53 SEQ ID NO: 8891 0.00% 0.9 20 85 SEQ ID NO: 8892 0.00% 0.9 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 196 SEQ ID NO: 8893 27.55% 440 2 44 SEQ ID NO: 8894 18.78% 300 3 36 SEQ ID NO: 8895 12.52% 200 4 92 SEQ ID NO: 8896 12.52% 200 5 109 SEQ ID NO: 8897 2.70% 43.2 6 25 SEQ ID NO: 8898 1.87% 30 7 93 SEQ ID NO: 8899 1.12% 18 8 12 SEQ ID NO: 8900 0.75% 12 9 123 SEQ ID NO: 8901 0.70% 11.2 10 7 SEQ ID NO: 8902 0.64% 10.368 11 17 SEQ ID NO: 8903 0.52% 8.4 12 139 SEQ ID NO: 8904 0.52% 8.4 13 193 SEQ ID NO: 8905 0.46% 7.5 14 6 SEQ ID NO: 8906 0.45% 7.2 15 19 SEQ ID NO: 8907 0.45% 7.2 16 110 SEQ ID NO: 8908 0.45% 7.2 17 114 SEQ ID NO: 8909 0.45% 7.2 18 210 SEQ ID NO: 8910 0.45% 7.2 19 46 SEQ ID NO: 8911 0.42% 6.72 20 52 SEQ ID NO: 8912 0.37% 6 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 92 SEQ ID NO: 8913 7.51% 120 2 42 SEQ ID NO: 8914 2.63% 42 3 109 SEQ ID NO: 8915 2.25% 36 4 23 SEQ ID NO: 8916 1.87% 30 5 34 SEQ ID NO: 8917 0.75% 12 6 6 SEQ ID NO: 8918 0.64% 10.368 7 45 SEQ ID NO: 8919 0.63% 10.08 8 196 SEQ ID NO: 8920 0.62% 10 9 44 SEQ ID NO: 8921 0.56% 9 10 40 SEQ ID NO: 8922 0.55% 8.8 11 62 SEQ ID NO: 8923 0.46% 7.5 12 193 SEQ ID NO: 8924 0.46% 7.5 13 18 SEQ ID NO: 8925 0.45% 7.2 14 113 SEQ ID NO: 8926 0.45% 7.2 15 56 SEQ ID NO: 8927 0.37% 6 16 176 SEQ ID NO: 8928 0.37% 6 17 16 SEQ ID NO: 8929 0.35% 5.6 18 138 SEQ ID NO: 8930 0.35% 5.6 19 127 SEQ ID NO: 8931 0.33% 5.28 20 36 SEQ ID NO: 8932 0.31% 5 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 13 SEQ ID NO: 8933 0.04% 1793.676528 2 87 SEQ ID NO: 8934 0.03% 1415.3832 3 24 SEQ ID NO: 8935 0.01% 618.0996816 4 19 SEQ ID NO: 8936 0.00% 223.23708 5 12 SEQ ID NO: 8937 0.00% 210.36400875 6 51 SEQ ID NO: 8938 0.00% 198.30859992 7 53 SEQ ID NO: 8939 0.00% 194.477328 8 88 SEQ ID NO: 8940 0.00% 180.58536756 9 106 SEQ ID NO: 8941 0.00% 169.74828 10 54 SEQ ID NO: 8942 0.00% 70.09848 11 59 SEQ ID NO: 8943 0.00% 43.42032 12 94 SEQ ID NO: 8944 0.00% 41.792058 13 20 SEQ ID NO: 8945 0.00% 37.46088108 14 63 SEQ ID NO: 8946 0.00% 35.73520902 15 22 SEQ ID NO: 8947 0.00% 20.5916435109 16 47 SEQ ID NO: 8948 0.00% 12.233222865 17 66 SEQ ID NO: 8949 0.00% 12.2199 18 56 SEQ ID NO: 8950 0.00% 11.486706 19 67 SEQ ID NO: 8951 0.00% 6.416172 20 117 SEQ ID NO: 8952 0.00% 5.827464 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 43 SEQ ID NO: 8953 0.10% 3977.8497792 2 24 SEQ ID NO: 8954 0.02% 836.2525104 3 51 SEQ ID NO: 8955 0.02% 815.616432 4 49 SEQ ID NO: 8956 0.01% 660.3245145 5 19 SEQ ID NO: 8957 0.00% 251.837856 6 59 SEQ ID NO: 8958 0.00% 159.9696 7 12 SEQ ID NO: 8959 0.00% 155.245377 8 45 SEQ ID NO: 8960 0.00% 141.1974531 9 21 SEQ ID NO: 8961 0.00% 117.22672269 10 53 SEQ ID NO: 8962 0.00% 84.55536 11 87 SEQ ID NO: 8963 0.00% 65.5671672 12 13 SEQ ID NO: 8964 0.00% 64.88888616 13 153 SEQ ID NO: 8965 0.00% 49.13352 14 178 SEQ ID NO: 8966 0.00% 26.082 15 18 SEQ ID NO: 8967 0.00% 24.802259691 16 116 SEQ ID NO: 8968 0.00% 21.5616168 17 65 SEQ ID NO: 8969 0.00% 20.77383 18 86 SEQ ID NO: 8970 0.00% 15.7068219 19 27 SEQ ID NO: 8971 0.00% 12.3159135 20 46 SEQ ID NO: 8972 0.00% 11.45624789925 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 4 SEQ ID NO: 8973 12.5% 4.5 2 136 SEQ ID NO: 8974 3.33% 1.2 3 156 SEQ ID NO: 8975 3.33% 1.2 4 140 SEQ ID NO: 8976 1.66% 0.6 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 169 SEQ ID NO: 8977 5.55% 2 2 94 SEQ ID NO: 8978 3.33% 1.2 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 146 SEQ ID NO: 8979 0.74% 40 2 154 SEQ ID NO: 8980 0.74% 40 3 80 SEQ ID NO: 8981 0.66% 36 4 139 SEQ ID NO: 8982 0.33% 18 5 83 SEQ ID NO: 8983 0.22% 12 6 209 SEQ ID NO: 8984 0.22% 12 7 7 SEQ ID NO: 8985 0.11% 6 8 3 SEQ ID NO: 8986 0.07% 4 9 6 SEQ ID NO: 8987 0.07% 4 10 12 SEQ ID NO: 8988 0.07% 4 11 19 SEQ ID NO: 8989 0.07% 4 12 24 SEQ ID NO: 8990 0.07% 4 13 38 SEQ ID NO: 8991 0.07% 4 14 46 SEQ ID NO: 8992 0.07% 4 15 56 SEQ ID NO: 8993 0.07% 4 16 110 SEQ ID NO: 8994 0.07% 4 17 114 SEQ ID NO: 8995 0.07% 4 18 123 SEQ ID NO: 8996 0.07% 4 19 129 SEQ ID NO: 8997 0.07% 4 20 166 SEQ ID NO: 8998 0.07% 4 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 56 SEQ ID NO: 8999 1.48% 80 2 40 SEQ ID NO: 9000 0.74% 40 3 127 SEQ ID NO: 9001 0.74% 40 4 170 SEQ ID NO: 9002 0.74% 40 5 140 SEQ ID NO: 9003 0.27% 15 6 35 SEQ ID NO: 9004 0.22% 12 7 79 SEQ ID NO: 9005 0.22% 12 8 82 SEQ ID NO: 9006 0.22% 12 9 208 SEQ ID NO: 9007 0.22% 12 10 209 SEQ ID NO: 9008 0.22% 12 11 80 SEQ ID NO: 9009 0.16% 9 12 129 SEQ ID NO: 9010 0.14% 8 13 138 SEQ ID NO: 9011 0.11% 6 14 73 SEQ ID NO: 9012 0.09% 5 15 2 SEQ ID NO: 9013 0.07% 4 16 5 SEQ ID NO: 9014 0.07% 4 17 6 SEQ ID NO: 9015 0.07% 4 18 16 SEQ ID NO: 9016 0.07% 4 19 18 SEQ ID NO: 9017 0.07% 4 20 24 SEQ ID NO: 9018 0.07% 4

TABLE 21 Epitopes for SEQ ID NO: 6047 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 53 SEQ ID NO: 9019 2% 112.5 2 10 SEQ ID NO: 9020 0.08% 4.5 3 33 SEQ ID NO: 9021 0.02% 1.5 4 3 SEQ ID NO: 9022 0.00% 0.5 5 27 SEQ ID NO: 9023 0.00% 0.5 6 29 SEQ ID NO: 9024 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 10 SEQ ID NO: 9025 0.8% 45 2 52 SEQ ID NO: 9026 0.2% 11.25 3 50 SEQ ID NO: 9027 0.04% 2.5 4 32 SEQ ID NO: 9028 0.02% 1.5 5 48 SEQ ID NO: 9029 0.02% 1.35 6 27 SEQ ID NO: 9030 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 38 SEQ ID NO: 9031 1.85% 225 2 17 SEQ ID NO: 9032 0.02% 3.6 3 2 SEQ ID NO: 9033 0.02% 2.7 4 37 SEQ ID NO: 9034 0.01% 1.8 5 27 SEQ ID NO: 9035 0.01% 1.35 6 13 SEQ ID NO: 9036 0.00% 0.675 7 14 SEQ ID NO: 9037 0.00% 0.6 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 13 SEQ ID NO: 9038 0.04% 6 2 37 SEQ ID NO: 9039 0.01% 2.025 3 2 SEQ ID NO: 9040 0.00% 0.9 4 19 SEQ ID NO: 9041 0.00% 0.675 5 16 SEQ ID NO: 9042 0.00% 0.54 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 20 SEQ ID NO: 9043 1.25% 20 2 6 SEQ ID NO: 9044 0.52% 8.4 3 5 SEQ ID NO: 9045 0.51% 8.25 4 35 SEQ ID NO: 9046 0.36% 5.76 5 31 SEQ ID NO: 9047 0.35% 5.6 6 43 SEQ ID NO: 9048 0.27% 4.4 7 13 SEQ ID NO: 9049 0.26% 4.2 8 32 SEQ ID NO: 9050 0.21% 3.36 9 2 SEQ ID NO: 9051 0.11% 1.8 10 9 SEQ ID NO: 9052 0.10% 1.68 11 8 SEQ ID NO: 9053 0.09% 1.5 12 15 SEQ ID NO: 9054 0.09% 1.5 13 23 SEQ ID NO: 9055 0.09% 1.5 14 27 SEQ ID NO: 9056 0.08% 1.4 15 24 SEQ ID NO: 9057 0.07% 1.2 16 7 SEQ ID NO: 9058 0.06% 1 17 17 SEQ ID NO: 9059 0.06% 1 18 10 SEQ ID NO: 9060 0.05% 0.9 19 39 SEQ ID NO: 9061 0.04% 0.792 20 47 SEQ ID NO: 9062 0.04% 0.792 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 5 SEQ ID NO: 9063 2.63% 42 2 34 SEQ ID NO: 9064 0.54% 8.64 3 30 SEQ ID NO: 9065 0.52% 8.4 4 19 SEQ ID NO: 9066 0.50% 8 5 50 SEQ ID NO: 9067 0.33% 5.28 6 12 SEQ ID NO: 9068 0.26% 4.2 7 31 SEQ ID NO: 9069 0.21% 3.36 8 26 SEQ ID NO: 9070 0.15% 2.52 9 8 SEQ ID NO: 9071 0.13% 2.1 10 22 SEQ ID NO: 9072 0.12% 2 11 23 SEQ ID NO: 9073 0.11% 1.8 12 6 SEQ ID NO: 9074 0.09% 1.5 13 14 SEQ ID NO: 9075 0.09% 1.5 14 16 SEQ ID NO: 9076 0.09% 1.5 15 7 SEQ ID NO: 9077 0.06% 1 16 48 SEQ ID NO: 9078 0.04% 0.75 17 0 SEQ ID NO: 9079 0.04% 0.72 18 9 SEQ ID NO: 9080 0.04% 0.72 19 47 SEQ ID NO: 9081 0.04% 0.66 20 39 SEQ ID NO: 9082 0.03% 0.6 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 15 SEQ ID NO: 9083 0.00% 14.1442686 2 27 SEQ ID NO: 9084 0.00% 9.598176 3 22 SEQ ID NO: 9085 0.00% 9.5634 4 9 SEQ ID NO: 9086 0.00% 5.546246013 5 2 SEQ ID NO: 9087 0.00% 5.526462816 6 24 SEQ ID NO: 9088 0.00% 4.88163753 7 17 SEQ ID NO: 9089 0.00% 3.699285408 8 31 SEQ ID NO: 9090 0.00% 2.29699206 9 6 SEQ ID NO: 9091 0.00% 2.0016040674 10 7 SEQ ID NO: 9092 0.00% 0.91287 11 49 SEQ ID NO: 9093 0.00% 0.71805678 12 16 SEQ ID NO: 9094 0.00% 0.6694257042 13 12 SEQ ID NO: 9095 0.00% 0.6539828625 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 16 SEQ ID NO: 9096 0.00% 34.28765802 2 19 SEQ ID NO: 9097 0.00% 18.9368775 3 14 SEQ ID NO: 9098 0.00% 14.1442686 4 27 SEQ ID NO: 9099 0.00% 11.406528 5 26 SEQ ID NO: 9100 0.00% 10.9304361558 6 34 SEQ ID NO: 9101 0.00% 5.580927 7 6 SEQ ID NO: 9102 0.00% 4.865742 8 9 SEQ ID NO: 9103 0.00% 2.64106953 9 50 SEQ ID NO: 9104 0.00% 2.6275752 10 30 SEQ ID NO: 9105 0.00% 2.29699206 11 7 SEQ ID NO: 9106 0.00% 0.86083641 12 42 SEQ ID NO: 9107 0.00% 0.7049592 13 22 SEQ ID NO: 9108 0.00% 0.6628440357 14 2 SEQ ID NO: 9109 0.00% 0.6530644656 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 37 SEQ ID NO: 9110 15% 5.4 2 38 SEQ ID NO: 9111 2.22% 0.8 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 37 SEQ ID NO: 9112 7.5% 2.7 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 35 SEQ ID NO: 9113 3.70% 200 2 17 SEQ ID NO: 9114 0.11% 6 3 6 SEQ ID NO: 9115 0.07% 4 4 20 SEQ ID NO: 9116 0.07% 4 5 31 SEQ ID NO: 9117 0.07% 4 6 43 SEQ ID NO: 9118 0.07% 4 7 7 SEQ ID NO: 9119 0.03% 2 8 23 SEQ ID NO: 9120 0.02% 1.2 9 24 SEQ ID NO: 9121 0.02% 1.2 10 10 SEQ ID NO: 9122 0.01% 0.9 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 35 SEQ ID NO: 9123 0.09% 5 2 19 SEQ ID NO: 9124 0.07% 4 3 30 SEQ ID NO: 9125 0.07% 4 4 34 SEQ ID NO: 9126 0.07% 4 5 7 SEQ ID NO: 9127 0.03% 2 6 16 SEQ ID NO: 9128 0.03% 1.8 7 23 SEQ ID NO: 9129 0.02% 1.2 8 50 SEQ ID NO: 9130 0.02% 1.2 9 9 SEQ ID NO: 9131 0.01% 1

TABLE 22 Epitopes for SEQ ID NO: 6048 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 66 SEQ ID NO: 9132 0.44% 25 2 80 SEQ ID NO: 9133 0.08% 5 3 93 SEQ ID NO: 9134 0.04% 2.7 4 11 SEQ ID NO: 9135 0.04% 2.5 5 89 SEQ ID NO: 9136 0.04% 2.25 6 48 SEQ ID NO: 9137 0.01% 1 7 3 SEQ ID NO: 9138 0.00% 0.5 8 9 SEQ ID NO: 9139 0.00% 0.5 9 56 SEQ ID NO: 9140 0.00% 0.5 10 101 SEQ ID NO: 9141 0.00% 0.5 11 106 SEQ ID NO: 9142 0.00% 0.5 12 110 SEQ ID NO: 9143 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 30 SEQ ID NO: 9144 0.4% 22.5 2 88 SEQ ID NO: 9145 0.12% 6.75 3 48 SEQ ID NO: 9146 0.04% 2.5 4 55 SEQ ID NO: 9147 0.02% 1.25 5 13 SEQ ID NO: 9148 0.01% 0.9 6 79 SEQ ID NO: 9149 0.01% 0.75 7 93 SEQ ID NO: 9150 0.01% 0.675 8 2 SEQ ID NO: 9151 0.00% 0.5 9 8 SEQ ID NO: 9152 0.00% 0.5 10 65 SEQ ID NO: 9153 0.00% 0.5 11 66 SEQ ID NO: 9154 0.00% 0.5 12 80 SEQ ID NO: 9155 0.00% 0.5 13 105 SEQ ID NO: 9156 0.00% 0.5 14 109 SEQ ID NO: 9157 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 109 SEQ ID NO: 9158 0.74% 90 2 3 SEQ ID NO: 9159 0.24% 30 3 111 SEQ ID NO: 9160 0.12% 15 4 106 SEQ ID NO: 9161 0.07% 9 5 95 SEQ ID NO: 9162 0.05% 6.075 6 101 SEQ ID NO: 9163 0.04% 6 7 110 SEQ ID NO: 9164 0.02% 3.6 8 84 SEQ ID NO: 9165 0.02% 3 9 80 SEQ ID NO: 9166 0.02% 2.7 10 37 SEQ ID NO: 9167 0.01% 2.25 11 9 SEQ ID NO: 9168 0.01% 2 12 54 SEQ ID NO: 9169 0.01% 2 13 99 SEQ ID NO: 9170 0.01% 1.35 14 1 SEQ ID NO: 9171 0.01% 1.215 15 11 SEQ ID NO: 9172 0.00% 0.9 16 15 SEQ ID NO: 9173 0.00% 0.9 17 69 SEQ ID NO: 9174 0.00% 0.6 18 5 SEQ ID NO: 9175 0.00% 0.54 19 103 SEQ ID NO: 9176 0.00% 0.54 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 75 SEQ ID NO: 9177 0.49% 60 2 109 SEQ ID NO: 9178 0.29% 36 3 22 SEQ ID NO: 9179 0.14% 18 4 15 SEQ ID NO: 9180 0.04% 6 5 110 SEQ ID NO: 9181 0.01% 2.25 6 95 SEQ ID NO: 9182 0.01% 1.8 7 101 SEQ ID NO: 9183 0.01% 1.35 8 43 SEQ ID NO: 9184 0.00% 1 9 2 SEQ ID NO: 9185 0.00% 0.9 10 5 SEQ ID NO: 9186 0.00% 0.9 11 7 SEQ ID NO: 9187 0.00% 0.9 12 107 SEQ ID NO: 9188 0.00% 0.9 13 102 SEQ ID NO: 9189 0.00% 0.81 14 3 SEQ ID NO: 9190 0.00% 0.75 15 8 SEQ ID NO: 9191 0.00% 0.6 16 103 SEQ ID NO: 9192 0.00% 0.54 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 88 SEQ ID NO: 9193 1.66% 26.6112 2 77 SEQ ID NO: 9194 0.77% 12.32 3 18 SEQ ID NO: 9195 0.56% 9 4 108 SEQ ID NO: 9196 0.56% 9 5 92 SEQ ID NO: 9197 0.54% 8.64 6 96 SEQ ID NO: 9198 0.54% 8.64 7 73 SEQ ID NO: 9199 0.46% 7.5 8 40 SEQ ID NO: 9200 0.45% 7.2 9 104 SEQ ID NO: 9201 0.42% 6.72 10 8 SEQ ID NO: 9202 0.41% 6.6 11 21 SEQ ID NO: 9203 0.37% 6 12 102 SEQ ID NO: 9204 0.37% 6 13 22 SEQ ID NO: 9205 0.25% 4 14 68 SEQ ID NO: 9206 0.25% 4 15 106 SEQ ID NO: 9207 0.22% 3.6 16 1 SEQ ID NO: 9208 0.18% 3 17 79 SEQ ID NO: 9209 0.18% 3 18 93 SEQ ID NO: 9210 0.18% 3 19 101 SEQ ID NO: 9211 0.18% 3 20 37 SEQ ID NO: 9212 0.15% 2.4 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 100 SEQ ID NO: 9213 0.93% 15 2 18 SEQ ID NO: 9214 0.78% 12.6 3 98 SEQ ID NO: 9215 0.52% 8.4 4 73 SEQ ID NO: 9216 0.46% 7.5 5 91 SEQ ID NO: 9217 0.45% 7.2 6 103 SEQ ID NO: 9218 0.42% 6.72 7 7 SEQ ID NO: 9219 0.41% 6.6 8 21 SEQ ID NO: 9220 0.37% 6 9 46 SEQ ID NO: 9221 0.37% 6 10 93 SEQ ID NO: 9222 0.37% 6 11 96 SEQ ID NO: 9223 0.37% 6 12 101 SEQ ID NO: 9224 0.37% 6 13 77 SEQ ID NO: 9225 0.25% 4 14 92 SEQ ID NO: 9226 0.22% 3.6 15 105 SEQ ID NO: 9227 0.22% 3.6 16 2 SEQ ID NO: 9228 0.18% 3 17 53 SEQ ID NO: 9229 0.18% 3 18 36 SEQ ID NO: 9230 0.12% 2 19 55 SEQ ID NO: 9231 0.12% 2 20 102 SEQ ID NO: 9232 0.11% 1.8 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 84 SEQ ID NO: 9233 0.01% 441.342216 2 102 SEQ ID NO: 9234 0.00% 63.16728165 3 107 SEQ ID NO: 9235 0.00% 51.882640425 4 1 SEQ ID NO: 9236 0.00% 43.8816609 5 95 SEQ ID NO: 9237 0.00% 33.40165248 6 2 SEQ ID NO: 9238 0.00% 24.66305226 7 92 SEQ ID NO: 9239 0.00% 22.64458905 8 103 SEQ ID NO: 9240 0.00% 20.70206586 9 47 SEQ ID NO: 9241 0.00% 11.175953184 10 94 SEQ ID NO: 9242 0.00% 8.452983 11 15 SEQ ID NO: 9243 0.00% 8.1793152 12 8 SEQ ID NO: 9244 0.00% 4.993461 13 5 SEQ ID NO: 9245 0.00% 4.57284528 14 99 SEQ ID NO: 9246 0.00% 3.999468528 15 105 SEQ ID NO: 9247 0.00% 2.231322 16 20 SEQ ID NO: 9248 0.00% 1.3524 17 62 SEQ ID NO: 9249 0.00% 0.8631693 18 6 SEQ ID NO: 9250 0.00% 0.824619 19 57 SEQ ID NO: 9251 0.00% 0.72105 20 58 SEQ ID NO: 9252 0.00% 0.7147572 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 101 SEQ ID NO: 9253 0.03% 1243.078056 2 3 SEQ ID NO: 9254 0.01% 592.944462 3 106 SEQ ID NO: 9255 0.00% 94.2678 4 5 SEQ ID NO: 9256 0.00% 43.42032 5 107 SEQ ID NO: 9257 0.00% 33.30332334 6 102 SEQ ID NO: 9258 0.00% 32.53181778 7 54 SEQ ID NO: 9259 0.00% 27.324 8 7 SEQ ID NO: 9260 0.00% 21.3624 9 1 SEQ ID NO: 9261 0.00% 13.723479 10 95 SEQ ID NO: 9262 0.00% 13.00344192 11 94 SEQ ID NO: 9263 0.00% 10.01276388 12 99 SEQ ID NO: 9264 0.00% 5.6615328 13 39 SEQ ID NO: 9265 0.00% 3.6304212 14 111 SEQ ID NO: 9266 0.00% 2.53368 15 103 SEQ ID NO: 9267 0.00% 2.475394803 16 14 SEQ ID NO: 9268 0.00% 2.4519012 17 19 SEQ ID NO: 9269 0.00% 2.07604992 18 29 SEQ ID NO: 9270 0.00% 1.8179154 19 57 SEQ ID NO: 9271 0.00% 1.52076 20 47 SEQ ID NO: 9272 0.00% 1.27712376 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 80 SEQ ID NO: 9273 3.33% 1.2 2 69 SEQ ID NO: 9274 1.66% 0.6 3 109 SEQ ID NO: 9275 1.66% 0.6 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 22 SEQ ID NO: 9276 11.11% 4 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 22 SEQ ID NO: 9277 3.70% 200 2 77 SEQ ID NO: 9278 2.22% 120 3 104 SEQ ID NO: 9279 0.22% 12 4 40 SEQ ID NO: 9280 0.11% 6 5 8 SEQ ID NO: 9281 0.07% 4 6 21 SEQ ID NO: 9282 0.07% 4 7 68 SEQ ID NO: 9283 0.07% 4 8 92 SEQ ID NO: 9284 0.07% 4 9 102 SEQ ID NO: 9285 0.07% 4 10 46 SEQ ID NO: 9286 0.03% 2 11 98 SEQ ID NO: 9287 0.03% 2 12 103 SEQ ID NO: 9288 0.03% 2 13 88 SEQ ID NO: 9289 0.02% 1.2 14 105 SEQ ID NO: 9290 0.01% 0.9 15 43 SEQ ID NO: 9291 0.01% 0.6 16 79 SEQ ID NO: 9292 0.01% 0.6 17 95 SEQ ID NO: 9293 0.01% 0.6 18 107 SEQ ID NO: 9294 0.00% 0.5 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 46 SEQ ID NO: 9295 1.48% 80 2 98 SEQ ID NO: 9296 1.48% 80 3 91 SEQ ID NO: 9297 0.37% 20 4 103 SEQ ID NO: 9298 0.37% 20 5 7 SEQ ID NO: 9299 0.07% 4 6 21 SEQ ID NO: 9300 0.07% 4 7 101 SEQ ID NO: 9301 0.07% 4 8 107 SEQ ID NO: 9302 0.03% 2 9 67 SEQ ID NO: 9303 0.02% 1.2 10 93 SEQ ID NO: 9304 0.02% 1.2 11 69 SEQ ID NO: 9305 0.01% 1 12 39 SEQ ID NO: 9306 0.01% 0.6 13 77 SEQ ID NO: 9307 0.01% 0.6 14 22 SEQ ID NO: 9308 0.00% 0.5

TABLE 23 Epitopes for SEQ ID NO: 6049 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 0 SEQ ID NO: 9309 0.2% 11.25 2 35 SEQ ID NO: 9310 0.01% 0.9 3 4 SEQ ID NO: 9311 0.00% 0.5 4 5 SEQ ID NO: 9312 0.00% 0.5 5 10 SEQ ID NO: 9313 0.00% 0.5 6 19 SEQ ID NO: 9314 0.00% 0.5 7 21 SEQ ID NO: 9315 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 0 SEQ ID NO: 9316 0.2% 11.25 2 5 SEQ ID NO: 9317 0.04% 2.5 3 33 SEQ ID NO: 9318 0.02% 1.5 4 3 SEQ ID NO: 9319 0.02% 1.25 5 9 SEQ ID NO: 9320 0.00% 0.5 6 18 SEQ ID NO: 9321 0.00% 0.5 7 20 SEQ ID NO: 9322 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 4 SEQ ID NO: 9323 0.14% 18 2 16 SEQ ID NO: 9324 0.11% 13.5 3 23 SEQ ID NO: 9325 0.06% 8.1 4 18 SEQ ID NO: 9326 0.03% 4.05 5 21 SEQ ID NO: 9327 0.01% 2.025 6 9 SEQ ID NO: 9328 0.01% 1.8 7 15 SEQ ID NO: 9329 0.01% 1.8 8 25 SEQ ID NO: 9330 0.01% 1.8 9 12 SEQ ID NO: 9331 0.00% 0.9 10 19 SEQ ID NO: 9332 0.00% 0.9 11 20 SEQ ID NO: 9333 0.00% 0.9 12 2 SEQ ID NO: 9334 0.00% 0.81 13 22 SEQ ID NO: 9335 0.00% 0.81 14 10 SEQ ID NO: 9336 0.00% 0.6 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 20 SEQ ID NO: 9337 0.16% 20.25 2 9 SEQ ID NO: 9338 0.09% 12 3 16 SEQ ID NO: 9339 0.07% 9 4 18 SEQ ID NO: 9340 0.07% 9 5 22 SEQ ID NO: 9341 0.06% 8.1 6 4 SEQ ID NO: 9342 0.03% 4.05 7 15 SEQ ID NO: 9343 0.03% 4.05 8 12 SEQ ID NO: 9344 0.02% 3.6 9 3 SEQ ID NO: 9345 0.00% 0.9 10 33 SEQ ID NO: 9346 0.00% 0.6 11 2 SEQ ID NO: 9347 0.00% 0.54 12 24 SEQ ID NO: 9348 0.00% 0.54 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 8 SEQ ID NO: 9349 18.78% 300 2 11 SEQ ID NO: 9350 1.87% 30 3 28 SEQ ID NO: 9351 1.50% 24 4 7 SEQ ID NO: 9352 0.75% 12 5 17 SEQ ID NO: 9353 0.56% 9 6 14 SEQ ID NO: 9354 0.46% 7.5 7 23 SEQ ID NO: 9355 0.37% 6 8 13 SEQ ID NO: 9356 0.36% 5.76 9 2 SEQ ID NO: 9357 0.35% 5.6 10 16 SEQ ID NO: 9358 0.35% 5.6 11 9 SEQ ID NO: 9359 0.30% 4.8 12 21 SEQ ID NO: 9360 0.26% 4.2 13 5 SEQ ID NO: 9361 0.25% 4 14 4 SEQ ID NO: 9362 0.22% 3.6 15 0 SEQ ID NO: 9363 0.18% 3 16 19 SEQ ID NO: 9364 0.18% 3 17 10 SEQ ID NO: 9365 0.15% 2.4 18 18 SEQ ID NO: 9366 0.13% 2.1 19 25 SEQ ID NO: 9367 0.06% 1.1 20 15 SEQ ID NO: 9368 0.05% 0.9 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 8 SEQ ID NO: 9369 22.54% 360 2 7 SEQ ID NO: 9370 1.25% 20 3 17 SEQ ID NO: 9371 0.65% 10.5 4 15 SEQ ID NO: 9372 0.52% 8.4 5 4 SEQ ID NO: 9373 0.45% 7.2 6 22 SEQ ID NO: 9374 0.37% 6 7 12 SEQ ID NO: 9375 0.36% 5.76 8 27 SEQ ID NO: 9376 0.30% 4.8 9 14 SEQ ID NO: 9377 0.28% 4.5 10 20 SEQ ID NO: 9378 0.26% 4.2 11 10 SEQ ID NO: 9379 0.25% 4 12 3 SEQ ID NO: 9380 0.18% 3 13 18 SEQ ID NO: 9381 0.18% 3 14 9 SEQ ID NO: 9382 0.15% 2.4 15 24 SEQ ID NO: 9383 0.10% 1.65 16 16 SEQ ID NO: 9384 0.07% 1.2 17 13 SEQ ID NO: 9385 0.06% 1 18 11 SEQ ID NO: 9386 0.05% 0.9 19 1 SEQ ID NO: 9387 0.05% 0.84 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 12 SEQ ID NO: 9388 0.10% 4267.988928 2 23 SEQ ID NO: 9389 0.03% 1360.69088544 3 9 SEQ ID NO: 9390 0.01% 569.948832 4 16 SEQ ID NO: 9391 0.00% 309.0498408 5 15 SEQ ID NO: 9392 0.00% 79.73570448 6 2 SEQ ID NO: 9393 0.00% 51.109542 7 18 SEQ ID NO: 9394 0.00% 45.25539984 8 25 SEQ ID NO: 9395 0.00% 34.28765802 9 22 SEQ ID NO: 9396 0.00% 26.532116325 10 5 SEQ ID NO: 9397 0.00% 25.26691266 11 21 SEQ ID NO: 9398 0.00% 4.72873208445 12 11 SEQ ID NO: 9399 0.00% 2.638538265 13 8 SEQ ID NO: 9400 0.00% 2.4274552038 14 4 SEQ ID NO: 9401 0.00% 1.7415324 15 20 SEQ ID NO: 9402 0.00% 1.6025526 16 13 SEQ ID NO: 9403 0.00% 1.453803297 17 35 SEQ ID NO: 9404 0.00% 1.36878336 18 3 SEQ ID NO: 9405 0.00% 0.824619 19 33 SEQ ID NO: 9406 0.00% 0.513774 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 22 SEQ ID NO: 9407 0.09% 3636.068421648 2 4 SEQ ID NO: 9408 0.02% 1107.960876 3 15 SEQ ID NO: 9409 0.02% 836.2525104 4 16 SEQ ID NO: 9410 0.00% 150.9313176 5 12 SEQ ID NO: 9411 0.00% 76.55002416 6 1 SEQ ID NO: 9412 0.00% 49.0273014 7 10 SEQ ID NO: 9413 0.00% 42.1638414747 8 20 SEQ ID NO: 9414 0.00% 9.29480508 9 24 SEQ ID NO: 9415 0.00% 9.2669346 10 13 SEQ ID NO: 9416 0.00% 7.96581954 11 21 SEQ ID NO: 9417 0.00% 5.051306761875 12 5 SEQ ID NO: 9418 0.00% 2.6941464 13 11 SEQ ID NO: 9419 0.00% 2.3839914 14 34 SEQ ID NO: 9420 0.00% 1.465422 15 2 SEQ ID NO: 9421 0.00% 0.70794 16 9 SEQ ID NO: 9422 0.00% 0.6513048 17 19 SEQ ID NO: 9423 0.00% 0.51882640425 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 33 SEQ ID NO: 9424 1.66% 0.6 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 13 SEQ ID NO: 9425 0.22% 12 2 2 SEQ ID NO: 9426 0.07% 4 3 9 SEQ ID NO: 9427 0.07% 4 4 16 SEQ ID NO: 9428 0.07% 4 5 23 SEQ ID NO: 9429 0.07% 4 6 5 SEQ ID NO: 9430 0.02% 1.2 7 15 SEQ ID NO: 9431 0.01% 1 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 4 SEQ ID NO: 9432 0.07% 4 2 10 SEQ ID NO: 9433 0.07% 4 3 12 SEQ ID NO: 9434 0.07% 4 4 15 SEQ ID NO: 9435 0.07% 4 5 22 SEQ ID NO: 9436 0.07% 4 6 13 SEQ ID NO: 9437 0.02% 1.2

TABLE 24 Epitopes for SEQ ID NO: 6050 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 47 SEQ ID NO: 9438 0.01% 0.75 2 21 SEQ ID NO: 9439 0.00% 0.5 3 53 SEQ ID NO: 9440 0.00% 0.5 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 16 SEQ ID NO: 9441 0.04% 2.5 2 71 SEQ ID NO: 9442 0.04% 2.5 3 47 SEQ ID NO: 9443 0.02% 1.5 4 62 SEQ ID NO: 9444 0.01% 0.9 5 20 SEQ ID NO: 9445 0.00% 0.5 6 38 SEQ ID NO: 9446 0.00% 0.5 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 54 SEQ ID NO: 9447 0.02% 2.7 2 17 SEQ ID NO: 9448 0.01% 2 3 3 SEQ ID NO: 9449 0.01% 1.8 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 22 SEQ ID NO: 9450 0.09% 12 2 16 SEQ ID NO: 9451 0.01% 2 3 54 SEQ ID NO: 9452 0.00% 0.9 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 70 SEQ ID NO: 9453 2.10% 33.6 2 7 SEQ ID NO: 9454 1.12% 18 3 60 SEQ ID NO: 9455 0.46% 7.5 4 54 SEQ ID NO: 9456 0.37% 6 5 14 SEQ ID NO: 9457 0.31% 5 6 19 SEQ ID NO: 9458 0.30% 4.8 7 47 SEQ ID NO: 9459 0.30% 4.8 8 12 SEQ ID NO: 9460 0.25% 4 9 15 SEQ ID NO: 9461 0.25% 4 10 67 SEQ ID NO: 9462 0.25% 4 11 21 SEQ ID NO: 9463 0.18% 3 12 37 SEQ ID NO: 9464 0.06% 1 13 27 SEQ ID NO: 9465 0.03% 0.5 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 14 SEQ ID NO: 9466 12.52% 200 2 7 SEQ ID NO: 9467 0.93% 15 3 11 SEQ ID NO: 9468 0.75% 12 4 60 SEQ ID NO: 9469 0.56% 9 5 18 SEQ ID NO: 9470 0.45% 7.2 6 46 SEQ ID NO: 9471 0.45% 7.2 7 53 SEQ ID NO: 9472 0.37% 6 8 69 SEQ ID NO: 9473 0.35% 5.6 9 66 SEQ ID NO: 9474 0.25% 4 10 20 SEQ ID NO: 9475 0.12% 2 11 47 SEQ ID NO: 9476 0.07% 1.2 12 36 SEQ ID NO: 9477 0.06% 1 13 26 SEQ ID NO: 9478 0.04% 0.75 14 70 SEQ ID NO: 9479 0.04% 0.72 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 54 SEQ ID NO: 9480 0.02% 881.199 2 26 SEQ ID NO: 9481 0.00% 95.013 3 61 SEQ ID NO: 9482 0.00% 93.69648 4 19 SEQ ID NO: 9483 0.00% 40.2894864 5 74 SEQ ID NO: 9484 0.00% 12.6684 6 35 SEQ ID NO: 9485 0.00% 10.34586 7 69 SEQ ID NO: 9486 0.00% 3.3704706 8 13 SEQ ID NO: 9487 0.00% 1.656 9 15 SEQ ID NO: 9488 0.00% 1.47537042 10 68 SEQ ID NO: 9489 0.00% 0.966 11 22 SEQ ID NO: 9490 0.00% 0.942678 12 12 SEQ ID NO: 9491 0.00% 0.7669695 13 36 SEQ ID NO: 9492 0.00% 0.52661835 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 61 SEQ ID NO: 9493 0.00% 93.69648 2 25 SEQ ID NO: 9494 0.00% 63.33035625 3 34 SEQ ID NO: 9495 0.00% 50.232 4 53 SEQ ID NO: 9496 0.00% 45.2838375 5 26 SEQ ID NO: 9497 0.00% 14.35752 6 27 SEQ ID NO: 9498 0.00% 2.8557858 7 17 SEQ ID NO: 9499 0.00% 2.3973222 8 36 SEQ ID NO: 9500 0.00% 1.798209 9 69 SEQ ID NO: 9501 0.00% 1.03521597 10 67 SEQ ID NO: 9502 0.00% 0.966 11 68 SEQ ID NO: 9503 0.00% 0.910938 12 11 SEQ ID NO: 9504 0.00% 0.7669695 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 17 SEQ ID NO: 9505 2.22% 0.8 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 16 SEQ ID NO: 9506 5.55% 2 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 27 SEQ ID NO: 9507 0.37% 20 2 54 SEQ ID NO: 9508 0.22% 12 3 70 SEQ ID NO: 9509 0.22% 12 4 67 SEQ ID NO: 9510 0.11% 6 5 12 SEQ ID NO: 9511 0.07% 4 6 15 SEQ ID NO: 9512 0.07% 4 7 19 SEQ ID NO: 9513 0.07% 4 8 49 SEQ ID NO: 9514 0.03% 2 9 69 SEQ ID NO: 9515 0.03% 1.8 10 47 SEQ ID NO: 9516 0.02% 1.2 11 5 SEQ ID NO: 9517 0.01% 1 12 9 SEQ ID NO: 9518 0.01% 1 13 35 SEQ ID NO: 9519 0.01% 1 14 37 SEQ ID NO: 9520 0.01% 0.6 15 68 SEQ ID NO: 9521 0.01% 0.6 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 69 SEQ ID NO: 9522 0.66% 36 2 53 SEQ ID NO: 9523 0.22% 12 3 5 SEQ ID NO: 9524 0.13% 7.5 4 66 SEQ ID NO: 9525 0.11% 6 5 11 SEQ ID NO: 9526 0.07% 4 6 27 SEQ ID NO: 9527 0.07% 4 7 46 SEQ ID NO: 9528 0.07% 4 8 18 SEQ ID NO: 9529 0.02% 1.2 9 9 SEQ ID NO: 9530 0.01% 1 10 26 SEQ ID NO: 9531 0.01% 1 11 25 SEQ ID NO: 9532 0.01% 0.75 12 17 SEQ ID NO: 9533 0.01% 0.6 13 36 SEQ ID NO: 9534 0.01% 0.6 14 68 SEQ ID NO: 9535 0.01% 0.6 15 35 SEQ ID NO: 9536 0.00% 0.5 16 42 SEQ ID NO: 9537 0.00% 0.5 17 73 SEQ ID NO: 9538 0.00% 0.5

TABLE 25 Epitopes for SEQ ID NO: 6052 Start % of max. Rank position Sequence score Score HLA A1 - 9 mers Maximum possible score using this molecule type 5625 1 365 SEQ ID NO: 9539 0.8% 45 2 397 SEQ ID NO: 9540 0.44% 25 3 229 SEQ ID NO: 9541 0.32% 18 4 103 SEQ ID NO: 9542 0.17% 10 5 338 SEQ ID NO: 9543 0.17% 10 6 251 SEQ ID NO: 9544 0.16% 9 7 79 SEQ ID NO: 9545 0.11% 6.25 8 119 SEQ ID NO: 9546 0.10% 6 9 361 SEQ ID NO: 9547 0.08% 5 10 60 SEQ ID NO: 9548 0.04% 2.25 11 101 SEQ ID NO: 9549 0.04% 2.25 12 278 SEQ ID NO: 9550 0.04% 2.25 13 23 SEQ ID NO: 9551 0.02% 1.25 14 164 SEQ ID NO: 9552 0.02% 1.25 15 165 SEQ ID NO: 9553 0.02% 1.25 16 295 SEQ ID NO: 9554 0.02% 1.25 17 172 SEQ ID NO: 9555 0.01% 0.9 18 0 SEQ ID NO: 9556 0.01% 0.75 19 311 SEQ ID NO: 9557 0.01% 0.75 20 78 SEQ ID NO: 9558 0.01% 0.625 HLA A1 - 10 mers Maximum possible score using this molecule type 5625 1 114 SEQ ID NO: 9559 1.11% 62.5 2 134 SEQ ID NO: 9560 0.8% 45 3 365 SEQ ID NO: 9561 0.8% 45 4 77 SEQ ID NO: 9562 0.66% 37.5 5 103 SEQ ID NO: 9563 0.44% 25 6 23 SEQ ID NO: 9564 0.22% 12.5 7 338 SEQ ID NO: 9565 0.17% 10 8 361 SEQ ID NO: 9566 0.17% 10 9 324 SEQ ID NO: 9567 0.11% 6.25 10 375 SEQ ID NO: 9568 0.11% 6.25 11 79 SEQ ID NO: 9569 0.04% 2.5 12 295 SEQ ID NO: 9570 0.04% 2.5 13 346 SEQ ID NO: 9571 0.04% 2.5 14 378 SEQ ID NO: 9572 0.03% 2 15 251 SEQ ID NO: 9573 0.03% 1.8 16 214 SEQ ID NO: 9574 0.02% 1.125 17 160 SEQ ID NO: 9575 0.01% 1 18 172 SEQ ID NO: 9576 0.01% 0.9 19 229 SEQ ID NO: 9577 0.01% 0.9 20 376 SEQ ID NO: 9578 0.01% 0.9 HLA A3 - 9 mers Maximum possible score using this molecule type 12150 1 229 SEQ ID NO: 9579 0.49% 60 2 361 SEQ ID NO: 9580 0.27% 33.75 3 330 SEQ ID NO: 9581 0.16% 20 4 218 SEQ ID NO: 9582 0.09% 12 5 338 SEQ ID NO: 9583 0.04% 6 6 352 SEQ ID NO: 9584 0.04% 6 7 103 SEQ ID NO: 9585 0.04% 5.4 8 291 SEQ ID NO: 9586 0.01% 2 9 241 SEQ ID NO: 9587 0.01% 1.8 10 290 SEQ ID NO: 9588 0.01% 1.8 11 316 SEQ ID NO: 9589 0.01% 1.8 12 222 SEQ ID NO: 9590 0.01% 1.35 13 266 SEQ ID NO: 9591 0.01% 1.35 14 53 SEQ ID NO: 9592 0.00% 1 15 100 SEQ ID NO: 9593 0.00% 0.9 16 138 SEQ ID NO: 9594 0.00% 0.9 17 240 SEQ ID NO: 9595 0.00% 0.9 18 119 SEQ ID NO: 9596 0.00% 0.675 19 44 SEQ ID NO: 9597 0.00% 0.6 20 161 SEQ ID NO: 9598 0.00% 0.6 HLA A3 - 10 mers Maximum possible score using this molecule type 12150 1 338 SEQ ID NO: 9599 0.49% 60 2 160 SEQ ID NO: 9600 0.32% 40 3 352 SEQ ID NO: 9601 0.24% 30 4 361 SEQ ID NO: 9602 0.18% 22.5 5 103 SEQ ID NO: 9603 0.13% 16.2 6 290 SEQ ID NO: 9604 0.07% 9 7 351 SEQ ID NO: 9605 0.07% 9 8 44 SEQ ID NO: 9606 0.04% 6 9 228 SEQ ID NO: 9607 0.03% 4.05 10 394 SEQ ID NO: 9608 0.02% 3 11 240 SEQ ID NO: 9609 0.02% 2.7 12 100 SEQ ID NO: 9610 0.01% 1.8 13 114 SEQ ID NO: 9611 0.01% 1.8 14 93 SEQ ID NO: 9612 0.01% 1.5 15 134 SEQ ID NO: 9613 0.01% 1.5 16 221 SEQ ID NO: 9614 0.01% 1.35 17 330 SEQ ID NO: 9615 0.00% 1.2 18 112 SEQ ID NO: 9616 0.00% 0.9 19 218 SEQ ID NO: 9617 0.00% 0.9 20 55 SEQ ID NO: 9618 0.00% 0.6 HLA A24 - 9 mers Maximum possible score using this molecule type 1596.672 1 345 SEQ ID NO: 9619 1.50% 24 2 306 SEQ ID NO: 9620 0.75% 12 3 222 SEQ ID NO: 9621 0.54% 8.64 4 111 SEQ ID NO: 9622 0.51% 8.25 5 159 SEQ ID NO: 9623 0.45% 7.2 6 219 SEQ ID NO: 9624 0.45% 7.2 7 283 SEQ ID NO: 9625 0.45% 7.2 8 266 SEQ ID NO: 9626 0.42% 6.72 9 56 SEQ ID NO: 9627 0.41% 6.6 10 131 SEQ ID NO: 9628 0.37% 6 11 214 SEQ ID NO: 9629 0.37% 6 12 297 SEQ ID NO: 9630 0.37% 6 13 86 SEQ ID NO: 9631 0.31% 5 14 122 SEQ ID NO: 9632 0.31% 5 15 48 SEQ ID NO: 9633 0.30% 4.8 16 105 SEQ ID NO: 9634 0.30% 4.8 17 213 SEQ ID NO: 9635 0.30% 4.8 18 323 SEQ ID NO: 9636 0.30% 4.8 19 338 SEQ ID NO: 9637 0.30% 4.8 20 399 SEQ ID NO: 9638 0.30% 4.8 HLA A24 - 10 mers Maximum possible score using this molecule type 1596.672 1 65 SEQ ID NO: 9639 0.93% 15 2 306 SEQ ID NO: 9640 0.75% 12 3 95 SEQ ID NO: 9641 0.66% 10.56 4 36 SEQ ID NO: 9642 0.60% 9.6 5 385 SEQ ID NO: 9643 0.50% 8 6 111 SEQ ID NO: 9644 0.46% 7.5 7 104 SEQ ID NO: 9645 0.45% 7.2 8 214 SEQ ID NO: 9646 0.45% 7.2 9 221 SEQ ID NO: 9647 0.45% 7.2 10 277 SEQ ID NO: 9648 0.45% 7.2 11 150 SEQ ID NO: 9649 0.37% 6 12 152 SEQ ID NO: 9650 0.37% 6 13 158 SEQ ID NO: 9651 0.37% 6 14 171 SEQ ID NO: 9652 0.37% 6 15 343 SEQ ID NO: 9653 037% 6 16 110 SEQ ID NO: 9654 0.34% 5.5 17 85 SEQ ID NO: 9655 0.31% 5 18 47 SEQ ID NO: 9656 0.30% 4.8 19 213 SEQ ID NO: 9657 0.30% 4.8 20 218 SEQ ID NO: 9658 0.30% 4.8 HLA A 0201 - 9 mers Maximum possible score using this molecule type 3925227.1 1 222 SEQ ID NO: 9659 0.03% 1267.10434728 2 226 SEQ ID NO: 9660 0.00% 69.552 3 316 SEQ ID NO: 9661 0.00% 50.232 4 351 SEQ ID NO: 9662 0.00% 31.24872 5 159 SEQ ID NO: 9663 0.00% 13.6235739 6 406 SEQ ID NO: 9664 0.00% 11.4264 7 165 SEQ ID NO: 9665 0.00% 8.14407 8 238 SEQ ID NO: 9666 0.00% 7.0518 9 138 SEQ ID NO: 9667 0.00% 5.112072 10 130 SEQ ID NO: 9668 0.00% 3.00547233 11 303 SEQ ID NO: 9669 0.00% 2.59578 12 157 SEQ ID NO: 9670 0.00% 2.412585 13 219 SEQ ID NO: 9671 0.00% 2.103255861 14 305 SEQ ID NO: 9672 0.00% 1.86369 15 158 SEQ ID NO: 9673 0.00% 1.646892 16 331 SEQ ID NO: 9674 0.00% 1.614048 17 399 SEQ ID NO: 9675 0.00% 1.442246832 18 324 SEQ ID NO: 9676 0.00% 1.319625 19 312 SEQ ID NO: 9677 0.00% 1.233099 20 262 SEQ ID NO: 9678 0.00% 0.966 HLA A 0201 - 10 mers Maximum possible score using this molecule type 3925227.1 1 221 SEQ ID NO: 9679 0.00% 309.0498408 2 112 SEQ ID NO: 9680 0.00% 98.26704 3 330 SEQ ID NO: 9681 0.00% 98.26704 4 158 SEQ ID NO: 9682 0.00% 36.31608 5 218 SEQ ID NO: 9683 0.00% 24.0754248 6 124 SEQ ID NO: 9684 0.00% 12.2199 7 55 SEQ ID NO: 9685 0.00% 10.467576 8 315 SEQ ID NO: 9686 0.00% 7.7274204 9 350 SEQ ID NO: 9687 0.00% 4.296699 10 405 SEQ ID NO: 9688 0.00% 4.286487 11 388 SEQ ID NO: 9689 0.00% 4.054785 12 322 SEQ ID NO: 9690 0.00% 3.883803 13 130 SEQ ID NO: 9691 0.00% 3.428691903 14 45 SEQ ID NO: 9692 0.00% 3.411230625 15 132 SEQ ID NO: 9693 0.00% 2.99943 16 410 SEQ ID NO: 9694 0.00% 2.63718 17 316 SEQ ID NO: 9695 0.00% 2.48686074 18 104 SEQ ID NO: 9696 0.00% 2.477311485 19 164 SEQ ID NO: 9697 0.00% 2.2011 20 282 SEQ ID NO: 9698 0.00% 2.16591 HLA A 1101 - 9 mers Maximum possible score using this molecule type 36 1 361 SEQ ID NO: 9699 16.66% 6 2 53 SEQ ID NO: 9700 2.77% 1 3 240 SEQ ID NO: 9701 1.66% 0.6 4 241 SEQ ID NO: 9702 1.66% 0.6 HLA A 1101 - 10 mers Maximum possible score using this molecule type 36 1 361 SEQ ID NO: 9703 16.66% 6 2 93 SEQ ID NO: 9704 8.33% 3 3 338 SEQ ID NO: 9705 3.33% 1.2 4 134 SEQ ID NO: 9706 2.77% 1 5 228 SEQ ID NO: 9707 2.5% 0.9 6 160 SEQ ID NO: 9708 2.22% 0.8 7 239 SEQ ID NO: 9709 1.66% 0.6 8 240 SEQ ID NO: 9710 1.66% 0.6 9 257 SEQ ID NO: 9711 1.66% 0.6 10 379 SEQ ID NO: 9712 1.66% 0.6 HLA B7 - 9 mers Maximum possible score using this molecule type 5400 1 105 SEQ ID NO: 9713 14.81% 800 2 66 SEQ ID NO: 9714 1.48% 80 3 93 SEQ ID NO: 9715 0.92% 50 4 257 SEQ ID NO: 9716 0.55% 30 5 323 SEQ ID NO: 9717 0.37% 20 6 211 SEQ ID NO: 9718 0.22% 12 7 219 SEQ ID NO: 9719 0.22% 12 8 403 SEQ ID NO: 9720 0.18% 10 9 343 SEQ ID NO: 9721 0.14% 8 10 12 SEQ ID NO: 9722 0.11% 6 11 113 SEQ ID NO: 9723 0.11% 6 12 48 SEQ ID NO: 9724 0.07% 4 13 56 SEQ ID NO: 9725 0.07% 4 14 150 SEQ ID NO: 9726 0.07% 4 15 153 SEQ ID NO: 9727 0.07% 4 16 159 SEQ ID NO: 9728 0.07% 4 17 213 SEQ ID NO: 9729 0.07% 4 18 216 SEQ ID NO: 9730 0.07% 4 19 222 SEQ ID NO: 9731 0.07% 4 20 283 SEQ ID NO: 9732 0.07% 4 HLA B7 - 10 mers Maximum possible score using this molecule type 5400 1 36 SEQ ID NO: 9733 1.48% 80 2 150 SEQ ID NO: 9734 1.48% 80 3 343 SEQ ID NO: 9735 1.48% 80 4 12 SEQ ID NO: 9736 1.11% 60 5 308 SEQ ID NO: 9737 1.11% 60 6 130 SEQ ID NO: 9738 0.37% 20 7 55 SEQ ID NO: 9739 0.22% 12 8 210 SEQ ID NO: 9740 0.22% 12 9 218 SEQ ID NO: 9741 0.22% 12 10 201 SEQ ID NO: 9742 0.18% 10 11 121 SEQ ID NO: 9743 0.14% 8 12 391 SEQ ID NO: 9744 0.13% 7.5 13 112 SEQ ID NO: 9745 0.11% 6 14 385 SEQ ID NO: 9746 0.11% 6 15 47 SEQ ID NO: 9747 0.07% 4 16 66 SEQ ID NO: 9748 0.07% 4 17 95 SEQ ID NO: 9749 0.07% 4 18 104 SEQ ID NO: 9750 0.07% 4 19 152 SEQ ID NO: 9751 0.07% 4 20 158 SEQ ID NO: 9752 0.07% 4

TABLE 26 Cloned sequences for E. coli expression DNA length Cloning ORF bp pET pGEX P28 537 NdeI/XhoI P65 1917 NheI/HindIII Nsp1A 2495 NheI/XhoI Nsp1B 2153 NdeI/XhoI Nsp1C 2612 NdeI/XhoI Nsp2A 431 NdeI/XhoI BamHI/XhoI Nsp2B 426 NdeI/XhoI BamHI/XhoI Nsp3 870 NdeI/XhoI Nsp4 249 NdeI/XhoI BamHI/XhoI Nsp5 594 NheI/XhoI Nsp6 339 NdeI/XhoI BamHI/XhoI Nsp7 417 NdeI/XhoI BamHI/XhoI Nsp9A 1385 NheI/XhoI Nsp9B 1409 NdeI/XhoI Nsp10 1803 NheI/XhoI Nsp11 1581 NdeI/XhoI Nsp12 1038 NdeI/HindIII Nsp13 897 NdeI/XhoI Spike (S1) 1946 NdeI/XhoI Spike (S2) 1598 NdeI/XhoI Spike (S1-S2) 3545 NdeI/XhoI HR1 287 NdeI/XhoI BamHI/XhoI HR2 146 NdeI/XhoI BamHI/XhoI ORF3Δ100 525 NdeI/XhoI ORF4 465 NdeI/XhoI Envelope (E) 231 NdeI/XhoI BamHI/XhoI Matrix (M)Δ100 366 NdeI/XhoI BamHI/XhoI ORF7Δ18 137 NdeI/XhoI BamHI/XhoI ORF8 369 NdeI/XhoI BamHI/XhoI ORF9 135 NdeI/XhoI BamHI/XhoI ORF10 120 NheI/XhoI BamHI/XhoI ORF11 255 NdeI/XhoI BamHI/XhoI Nucleocapsid (N) 1269 NdeI/EcoRI ORF12 297 NdeI/EcoRI BamHI/EcoRI

TABLE 27 Primers ORF Forward primer Reverse primer P28 9803 9818 P65 9804 9819 Nsp1A 9805 9820 Nsp1B 9806 9821 Nsp1C 9807 9822 Nsp2 + Nsp3 9808 9823 Nsp4 to Nsp7 9809 9824 Nsp9A 9810 9825 Nsp9B 9811 9826 Nsp10 9812 9827 Nsp11 9813 9828 Nsp12-Nsp13 9814 9829 ORF3-ORF4 9815 9830 Env-ORF10 9816 9831 ORF11-ORF12 9817 9832

TABLE 28 Primers ORF Forward primer Reverse primer Nsp2A SEQ ID NO: 9833 SEQ ID NO: 9858 Nsp2B SEQ ID NO: 9834 SEQ ID NO: 9859 Nsp3 SEQ ID NO: 9835 SEQ ID NO: 9860 Nsp4 SEQ ID NO: 9836 SEQ ID NO: 9861 Nsp5 SEQ ID NO: 9837 SEQ ID NO: 9862 Nsp6 SEQ ID NO: 9838 SEQ ID NO: 9863 Nsp7 SEQ ID NO: 9839 SEQ ID NO: 9864 Nsp12 SEQ ID NO: 9840 SEQ ID NO: 9865 Nsp13 SEQ ID NO: 9841 SEQ ID NO: 9866 Spike S1 SEQ ID NO: 9842 SEQ ID NO: 9867 Spike S2 SEQ ID NO: 9843 SEQ ID NO: 9868 Spike S1-S2 SEQ ID NO: 9844 SEQ ID NO: 9869 HR1 SEQ ID NO: 9845 SEQ ID NO: 9870 HR2 SEQ ID NO: 9846 SEQ ID NO: 9871 Orf3Δ100 SEQ ID NO: 9847 SEQ ID NO: 9872 Orf4 SEQ ID NO: 9848 SEQ ID NO: 9873 Env E SEQ ID NO: 9849 SEQ ID NO: 9874 Matrix MΔ100 SEQ ID NO: 9850 SEQ ID NO: 9875 Orf7Δ18 SEQ ID NO: 9851 SEQ ID NO: 9876 Orf8 SEQ ID NO: 9852 SEQ ID NO: 9877 Orf9 SEQ ID NO: 9853 SEQ ID NO: 9878 Orf10 SEQ ID NO: 9854 SEQ ID NO: 9879 Orf11 SEQ ID NO: 9855 SEQ ID NO: 9880 Nucleocapsid N SEQ ID NO: 9856 SEQ ID NO: 9881 Orf12 SEQ ID NO: 9857 SEQ ID NO: 9882

TABLE 29 Cloning, purification and expression in E. coli M.W SARS CoV ORFs Kd cloning Expr. purification as P28 19.7 + + his sol P65 70.3 + + his sol Nsp1A (N-term) 91.6 + + his sol Nsp1B (core) 80.8 + − Nsp1C (C-term) 95.3 + − Nsp2A (N-term) 15.8 + + his ins Nsp2B (C-term) 15.5 + + his sol Nsp3 31.9 + − Nsp4 9.1 + + his sol Nsp5 21.8 + + his sol Nsp6 12.4 + + his sol Nsp7 15.3 + + his ins Nsp9A (N-term) 50.8 + − Nsp9B (C-term) 51.6 + + his ins Nsp10 66 − Nsp11 58 − Nsp12 38 − Nsp13 32.7 + + his ins Spike (S1-his) 71.3 + + his ins Spike (S2-his) 58.6 + − Spike (S1S2-his) 130 + + his ins HR1 11 + + his ins HR2 5.4 + + his sol ORF3 Δ100¹ 19.1 + − ORF4 16.9 + + his ins (trimer) Envelope (E) 34.3 + + gst ins (IB) Matrix (M) Δ100 13.3 + + his ins ORF7Δ18² 31 + + gst sol ORF8 39.5 + + gst ins (IB) ORF9 30.8 + + gst sol ORF10 30.3 + + gst ins (IB) ORF11 35.2 + + gst ins (IB) Nucleocapsid (N) 43.6 + + his ins ORF12 36.7 + + his ins

TABLE 30 E. coli expression, purity and yield Protein Tag Purity (%) Yield (mg/l) Nsp2A (N-term) His 95 1.7 Nsp2B (C-term) His 95 4.1 Nsp4 His 95 12.6 Nsp5 His 95 5.88 Nsp6 His 95 8.1 P28 His 95 1 P65 His 80 0.553 HR2 His 95 11.9 HR1 His 80 2.64 Nsp1A His 95 0.267 Spike S1-S2 His 80 0.381 Matrix M His 85 12.4 ORF7 GST 85 4.9

TABLE 31 Primers SEQ ID NO: Rank Model Local (Position) 10235 F1 1 1 (106) 10236 F2 2 1 (728) 10237 F3 3 1 (112) 10238 F4 5 2 (1331) 10239 F5 6 1 (12) 10240 F6 6 1 (346) 10241 F7 8 1 (904) 10242 F8 9 1 (1016) 10243 F9 9 1 (1015) 10244 F10 9 1 (719) 10245 F11 9 1 (720) 10246 F12 10 1 (724) 10247 R1 2 1 (1283) 10248 R2 4 1 (756) 10249 R3 4 1 (758) 10250 R4 5 2 (259) 10251 R5 6 1 (54) 10252 R6 7 1 (648) 10253 R7 8 1 (948) 10254 R8 8 1 (260) 10255 R9 9 1 (1282) 10256 R10 9 1 (950) 10257 R11 9 1 (756) 10258 R12 10 1 (132)

TABLE 32 Primers Scores Rank Model Local Sequence (Position) Primers List: (forward) F1 7 1 SEQ ID NO: 10352 (290) F2 7 1 SEQ ID NO: 10353 (291) F3 7 1 SEQ ID NO: 10354 (294) F4 7 1 SEQ ID NO: 10355 (292) F5 7 1 SEQ ID NO: 10356 (293) F6 9 1 SEQ ID NO: 10357 (198) F7 9 1 SEQ ID NO: 10358 (199) F8 10 1 SEQ ID NO: 10359 (33) F9 11 1 SEQ ID NO: 10360 (200) F10 11 1 SEQ ID NO: 10361 (299) F11 12 1 SEQ ID NO: 10362 (298) F12 12 1 SEQ ID NO: 10363 (297) F13 14 1 SEQ ID NO: 10364 (35) F14 14 1 SEQ ID NO: 10365 (34) F15 16 1 SEQ ID NO: 10366 (300) F16 17 1 SEQ ID NO: 10367 (295) F17 17 1 SEQ ID NO: 10368 (296) F18 17 1 SEQ ID NO: 10369 (175) F19 17 1 SEQ ID NO: 10370 (36) F20 20 1 SEQ ID NO: 10371 (202) F21 20 1 SEQ ID NO: 10372 (201) F22 28 1 SEQ ID NO: 10373 (204) F23 28 1 SEQ ID NO: 10374 (203) F24 29 1 SEQ ID NO: 10375 (269) F25 29 1 SEQ ID NO: 10376 (268) Primers List: (reverse) R1 7 1 SEQ ID NO: 10377 (337) R2 9 1 SEQ ID NO: 10378 (229) R3 11 1 SEQ ID NO: 10379 (230) R4 11 1 SEQ ID NO: 10380 (338) R5 12 1 SEQ ID NO: 10381 (207) R6 12 1 SEQ ID NO: 10382 (338) R7 13 1 SEQ ID NO: 10383 (231) R8 14 1 SEQ ID NO: 10384 (80) R9 14 1 SEQ ID NO: 10385 (232) R10 15 1 SEQ ID NO: 10386 (82) R11 16 1 SEQ ID NO: 10387 (340) R12 17 1 SEQ ID NO: 10388 (83) R13 17 1 SEQ ID NO: 10389 (206) R14 17 1 SEQ ID NO: 10390 (82) R15 17 1 SEQ ID NO: 10391 (337) R16 18 1 SEQ ID NO: 10392 (341) R17 20 1 SEQ ID NO: 10393 (340) R18 20 1 SEQ ID NO: 10394 (233) R19 21 1 SEQ ID NO: 10395 (79) R20 22 1 SEQ ID NO: 10396 (213) R21 28 1 SEQ ID NO: 10397 (236) R22 29 1 SEQ ID NO: 10398 (317) R23 32 1 SEQ ID NO: 10399 (391) R24 35 1 SEQ ID NO: 10400 (57) R25 36 1 SEQ ID NO: 10401 (237) Primers List (left part): SEQ ID NO^(S): 10402-10433 Primers List (right part): SEQ ID NO^(S): 10434-10464 Primers List (forward): SEQ ID NO^(S): 10465-10484 Primers List (reverse): SEQ ID NO^(S): 10485-10504

TABLE 33 Primers Scores Rank Model Local Sequence (Position) Primers List: (forward) F1 1 1 SEQ ID NO: 10580 (637) F2 2 1 SEQ ID NO: 10581 (439) F3 2 1 SEQ ID NO: 10582 (440) F4 3 1 SEQ ID NO: 10583 (729) F5 4 1 SEQ ID NO: 10584 (696) F6 4 1 SEQ ID NO: 10585 (697) F7 4 1 SEQ ID NO: 10586 (111) F8 5 1 SEQ ID NO: 10587 (867) F9 5 1 SEQ ID NO: 10588 (868) F10 5 1 SEQ ID NO: 10589 (869) F11 5 1 SEQ ID NO: 10590 (640) F12 6 1 SEQ ID NO: 10591 (438) F13 6 1 SEQ ID NO: 10592 (437) F14 6 1 SEQ ID NO: 10593 (436) F15 6 1 SEQ ID NO: 10594 (732) F16 6 1 SEQ ID NO: 10595 (635) F17 6 1 SEQ ID NO: 10596 (457) F18 6 1 SEQ ID NO: 10597 (458) F19 6 1 SEQ ID NO: 10598 (636) F20 7 1 SEQ ID NO: 10599 (854) F21 7 1 SEQ ID NO: 10600 (855) F22 7 1 SEQ ID NO: 10601 (581) F23 7 1 SEQ ID NO: 10602 (853) F24 7 1 SEQ ID NO: 10603 (342) F25 7 1 SEQ ID NO: 10604 (343) F26 7 1 SEQ ID NO: 10605 (112) F27 7 1 SEQ ID NO: 10606 (94) F28 7 1 SEQ ID NO: 10607 (642) F29 8 1 SEQ ID NO: 10608 (638) F30 8 1 SEQ ID NO: 10609 (639) F31 8 1 SEQ ID NO: 10610 (730) F32 8 1 SEQ ID NO: 10611 (641) F33 8 1 SEQ ID NO: 10612 (731) F34 8 1 SEQ ID NO: 10613 (326) F35 8 1 SEQ ID NO: 10614 (325) F36 9 1 SEQ ID NO: 10615 (517) F37 9 1 SEQ ID NO: 10616 (701) F38 9 1 SEQ ID NO: 10617 (208) F39 9 1 SEQ ID NO: 10618 (209) F40 9 1 SEQ ID NO: 10619 (702) F41 9 1 SEQ ID NO: 10620 (210) F42 10 1 SEQ ID NO: 10621 (634) F43 10 1 SEQ ID NO: 10622 (694) F44 10 1 SEQ ID NO: 10623 (693) F45 10 1 SEQ ID NO: 10624 (728) F46 10 1 SEQ ID NO: 10625 (695) F47 10 1 SEQ ID NO: 10626 (95) F48 11 1 SEQ ID NO: 10627 (455) F49 11 1 SEQ ID NO: 10628 (456) F50 11 1 SEQ ID NO: 10629 (454) Primers List: (reverse) R1 1 1 SEQ ID NO: 10630 (367) R2 1 1 SEQ ID NO: 10631 (666) R3 2 1 SEQ ID NO: 10632 (464) R4 3 1 SEQ ID NO: 10633 (669) R5 3 1 SEQ ID NO: 10634 (750) R6 4 1 SEQ ID NO: 10635 (720) R7 4 1 SEQ ID NO: 10636 (465) R8 4 1 SEQ ID NO: 10637 (370) R9 4 1 SEQ ID NO: 10638 (668) R10 4 1 SEQ ID NO: 10639 (135) R11 5 1 SEQ ID NO: 10640 (901) R12 5 1 SEQ ID NO: 10641 (667) R13 6 1 SEQ ID NO: 10642 (609) R14 6 1 SEQ ID NO: 10643 (464) R15 6 1 SEQ ID NO: 10644 (665) R16 6 1 SEQ ID NO: 10645 (486) R17 6 1 SEQ ID NO: 10646 (356) R18 6 1 SEQ ID NO: 10647 (758) R19 7 1 SEQ ID NO: 10648 (366) R20 7 1 SEQ ID NO: 10649 (368) R21 7 1 SEQ ID NO: 10650 (136) R22 7 1 SEQ ID NO: 10651 (675) R23 7 1 SEQ ID NO: 10652 (366) R24 7 1 SEQ ID NO: 10653 (608) R25 7 1 SEQ ID NO: 10654 (884) R26 7 1 SEQ ID NO: 10655 (120) R27 8 1 SEQ ID NO: 10656 (355) R28 8 1 SEQ ID NO: 10657 (671) R29 8 1 SEQ ID NO: 10658 (756) R30 8 1 SEQ ID NO: 10659 (751) R31 8 1 SEQ ID NO: 10660 (666) R32 9 1 SEQ ID NO: 10661 (242) R33 9 1 SEQ ID NO: 10662 (543) R34 9 1 SEQ ID NO: 10663 (724) R35 9 1 SEQ ID NO: 10664 (482) R36 10 1 SEQ ID NO: 10665 (121) R37 10 1 SEQ ID NO: 10666 (662) R38 10 1 SEQ ID NO: 10667 (750) R39 10 1 SEQ ID NO: 10668 (719) R40 10 1 SEQ ID NO: 10669 (242) R41 11 1 SEQ ID NO: 10670 (484) R42 11 1 SEQ ID NO: 10671 (375) R43 11 1 SEQ ID NO: 10672 (728) R44 11 1 SEQ ID NO: 10673 (373) R45 11 1 SEQ ID NO: 10674 (998) R46 11 1 SEQ ID NO: 10675 (486) R47 12 1 SEQ ID NO: 10676 (881) R48 12 1 SEQ ID NO: 10677 (882) R49 12 1 SEQ ID NO: 10678 (244) R50 12 1 SEQ ID NO: 10679 (1003) Primers List (left part): SEQ ID NO^(S): 10680-10974 Primers List (right part): SEQ ID NO^(S): 10975-11282 Primers List (forward): SEQ ID NO^(S): 11283-11302 Primers List (reverse): SEQ ID NO^(S): 11303-11322

TABLE 34 Compound # Structure Name MH+ 1

N-methyl-4-[(2-{[2-(1- methylethyl)phenyl]amino}-1H- benzimidazol-5-yl)oxy]pyridine- 2-carboxamide 402.5 2

N-methyl-4-{[1-methyl-2-({3- [(trimethylsilyl)ethynyl]phenyl}amino)-1H-benzimidazol-5- yl]oxy}pyridine-2-carboxamide 470.6 3

N-methyl-4-[(1-methyl-2-{[2- (phenylcarbonyl)phenyl]amino}- 1H-benzimidazol-5- yl)oxy]pyridine-2-carboxamide 478.5 4

4-(methyloxy)-N-[6- (methyloxy)-1,3-benzothiazol-2- yl]-3-nitrobenzamide 360.4 5

4-({2-[(4-butylphenyl)amino]- 1,3-benzothiazol-5-yl}oxy)-N- methylpyridine-2-carboxamide 433.5 6

N-methyl-4-({1-methyl-2-[(6- pyrrolidin-1-ylpyridin-3-yl) amino]-1H-benzimidazol-5- yl}oxy)pyridine-2-carboxamide 444.5 7

4-({2-[1,1′-bi(cyclohexyl)-2- ylamino]-1-methyl-1H- benzimidazol-5-yl}oxy)-N- methylpyridine-2-carboxamide 462.6 8

4-({2-[(4-chlorophenyl)amino]- 1-methyl-1H-benzimidazol-5- yl}oxy)-N-1,3-thiazol-2- ylpyridine-2-carboxamide 477.9 9

4-[(1-methyl-2-{[2-(methyloxy) phenyl]amino}-1H- benzimidazol-5-yl)oxy]-N-[3- (methyloxy)propyl]pyridine-2- carboxamide 462.5 10

4-({2-[(4-ethylphenyl)amino]- 1,3-benzoxazol-5-yl}oxy)-N- methylpyridine-2-carboxamide 389.4 11

1-[(3-fluorophenyl)carbonyl]-4- {[4-(trifluoromethyl)phenyl]methyl}pierazine 367.4 12

1-[2-(ethyloxy)phenyl]-4- {[3,4,5-tris(methyloxy)phenyl]carbonyl}piperazine 401.5 13

1-(3-chlorophenyl)-4-{[2- (ethyloxy)phenyl]carbonyl}piperazine 345.8 14

3-({4-[(2E)-3-phenylprop-2- enyl]piperazin-1-yl}carbonyl)-7- oxabicyclo[2.2.1]heptane-2- carboxylic acid 371.4 15

1-[2-(methyloxy)phenyl]-4- {[3,4,5-tris(methyloxy) phenyl]carbonyl}piperazine 387.4 16

3-[(4-pyridin-2-ylpiperazin-1- yl)carbonyl]-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid 332.4 17

3-pentyl-7-[(4-phenylpiperazin- 1-yl)carbonyl]-2-thioxo-2,3- dihydroquinazolin-4(1H)-one 437.6 18

1-[(E)-({4-[(2,4-dimethyl- phenyl)methyl]piperazin-1-yl}imino)methyl]naphthalen-2-ol 374.5 19

5-chloro-1-{[3- (trifluoromethyl)phenyl]methyl)-1H-indole-2,3-dione 340.7 20

1-[(4-methylphenyl)methyl]-5- nitro-1H-indole-2,3-dione 297.3 21

1-methyl-6,7-bis(methyloxy)-2- {[3-(methyloxy)phenyl]carbonyl}-1,2,3,4- tetrahydroisoquinoline 342.4 22

1-methyl-6,7-bis(methyloxy)-2- (naphthalen-2-ylcarbonyl)- 1,2,3,4-tetrahydroisoquinoline 362.4 23

[2-(trifluoromethyl)phenyl]methyl 3-[4-(aminocarbonyl) phenyl]-2-cycloheptyl-1-oxo- 1,2,3,4-tetrahydroisoquinoline- 4-carboxylate 565.6 24

anthra[1,2,5]thiadiazole- 6,11-dione 267.3 25

benzo[b]oxanthrene-6,11-dione 265.2 26

ethyl 6,11-dioxo-6,11- dihydrobenzo[b]phenazine-2- carboxylate 333.3 27

N,N-dimethyl-9,10-dioxo-9,10- dihydroanthracene-1- sulfonamide 316.3 28

2-(trifluoromethyl)-3-{[3,4,5- tris(methyloxy)phenyl]carbonyl}naphtho[2,3-b]furan-4,9-dione 461.4 29

2-(2-oxopropyl)-2-phenyl-1H- indene-1,3(2H)-dione 279.3 30

ethyl 4-{5-[(3- nitrophenyl)carbonyl]-1,3- dioxo-1,3-dihydro-2H-isoindol- 2-yl}benzoate 445.4 31

5,6-dichloro-2-[2-chloro-5- (trifluoromethyl)phenyl]-1H- isoindole-1,3(2H)-dione 395.6 32

3-bromo-4-{[(2- fluorophenyl)methyl]oxy}-5- (methyloxy)benzaldehyde thiosemicarbazone 413.3 33

2-[4-(3-chlorophenyl)piperazin- 1-yl]-5-nitrobenzaldehyde thiosemicarbazone 419.9 34

4-{[2-(3-chlorophenyl) ethyl]amino}-3- nitrobenzaldehyde thiosemicarbazone 378.9 35

(1E)-6,9-dimethyl-2,3,4,9- tetrahydro-1H-carbazol-1-one thiosemicarbazone 287.4 36

(2E)-1,1′-bi(cyclohexan)-1-en-2- one thiosemicarbazone 252.4 37

4-{[2-(4-chlorophenyl) ethyl]amino}-3-nitro- benzaldehyde thiosemicarbazone 378.9 38

4-(diethylamino)-2-{[(4- fluorophenyl)methyl]oxy}benzaldhyde N-(2-piperidin-1- ylethyl)thiosemicarbazone 486.7 39

3,4-bis(methyloxy)benzaldehyde (1,1-dioxido-1,2-benzisothiazol- 3-yl)(methyl)hydrazone 360.4 40

(2E)-2-[(4-chlorophenyl)(5- chlorothien-2-yl)methylidene]hydrazinecarboximidamide 314.2 41

2-(4-amino-2-oxo-1-propyl-1,2- dihydroquinolin-3-yl)-1H- benzimidazole-6-carbonitrile 344.4 42

4-amino-6-fluoro-7-({[4- (methyloxy)phenyl]methyl}amino)-3-[5-(4-methylpiperazin- 1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-one 528.6 43

6-chloro-3-(5-chloro-1H- benzimidazol-2-yl)-4-{[2- (dimethylamino)ethyl]amino}quinolin-2(1H)-one 417.3 44

4-amino-5-(1H-benzimidazol-2- yl)-1-methyl-1,7-dihydro-6H- pyrazolo[3,4-b]pyridin-6-one 281.3 45

5,5-dimethyl-4-methylidene-3- (2,4,6-trinitrophenyl)-1,3- oxazolidin-2-one 339.2 46

5-methyl-2-[4- (methyloxy)phenyl]hexahydro- 1H-isoindole-1,3(2H)-dione 274.3 47

5-methyl-2-(4- methylphenyl)hexahydro-1H- isoindole-1,3(2H)-dione 258.3 48

N-2˜-(4-chlorophenyl)-6,6- dimethyl-1,6-dihydro-1,3,5- triazine-2,4-diamine 252.7 49

(7Z)-7-(furan-2-ylmethylidene)- 3-phenyl-3,4-dihydro-2H- [1,3]thiazolo[3,2-a][1,3,5]triazin-6(7H)-one 312.4 50

(3aR,9R,9aR)-6,7-dihydroxy-9- [3,4,5-tris(methyloxy)phenyl]- 3a,4,9,9a-tetrahydronaphtho[2,3- c]furan-1(3H)-one 387.4 51

6-chloro-2-(ethyloxy)-4-methyl- 3-(4-nitrophenyl)-3a,4,9,9a- tetrahydro-3H-pyrrolo[2,3- b]quinoxaline 387.8 52

ethyl 2-(ethyloxy)-4-methyl- 3a,4,9,9a-tetrahydro-3H- pyrrolo[2,3-b]quinoxaline-3- carboxylate 304.4 53

ethyl 4-({[2,5-bis(methyloxy) phenyl]amino}methyl)- 3,5-dimethyl-1H-pyrrole-2- carboxylate 333.4 54

1-{3-[(6-amino-5-nitropyridin-2- yl)amino]propyl}-4-(2- chlorophenyl)-N-[(2S)-2- hydroxypropyl]-1H-pyrrole-3- carboxamide 473.9 55

(4-methylphenyl)(5-nitro-2- piperidin-1-ylphenyl)methanone 325.4 56

(2S,5R)-N˜1˜-(4-methyl- phenyl)-5-phenyl-N˜2˜-(2- pyridin-2-ylethyl)pyrrolidine- 1,2-dicarboxamide 429.5 57

2-[(3S)-3-(acetylamino)-2- oxopyrrolidin-1-yl]-N-[2-(4- fluorophenyl)ethyl]acetamide 322.4 58

N-[2-(2,4-dichlorophenyl)ethyl]- 4-({(Z)-[(4,4-difluoro- cyclohexyl)imino][(3S)-3- methylpiperazin-1- yl]methyl}amino)benzamide 553.5 59

4-[4-(methyloxy)phenyl]-5- phenylisoxazole 252.3 60

methyl 4-{[4-(1-methylethyl)- 2,3-dioxo-7-(trifluoromethyl)- 3,4-dihydroquinoxalin-1(2H)- yl]methyl}benzoate 421.4 61

(3beta, 16beta)-3,14,16- trihydroxybufa-20,22-dienolide 403.5 62

2-(aminomethyl)-1-(2-pyridin-2- ylethyl)quinazolin-4(1H)-one 281.3 63

ethyl 4-{[5-[3,4-bis(methyloxy) phenyl]-7-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3- yl]carbonyl}piperazine-1- carboxylate 508.5 64

5-[3,4-bis(methyloxy)phenyl]-3- (piperidin-1-ylcarbonyl)-7- (trifluoromethyl)pyrazolo[1,5- a]pyrimidine 435.4 65

5-[3,4-bis(methyloxy)phenyl]-N- methyl-N-(2-pyridin-2-ylethyl)- 7-(trifluoromethyl)pyrazolo[1,5- a]pyrimidine-2-carboxamide 486.5 66

5-propyl-2-thien-2- ylpyrazolo[1,5-a]pyrimidin-7-ol 260.3

BRIEF DESCRIPTION OF SEQUENCE LISTING SEQ ID NO: Description   1 Draft genome assembly from The Genome Science Center in British Colombia, Canada of sequence from TOR2 isolate. TOR2_draft_genome_assembly_120403 Release 1   2 CDC SARS-CoV strain sequence. Entire nucleotide sequence (Urbani strain)  3-20 Group-specific coronavirus gene products Feline infectious peritonitis virus (FIPV) 3/4 = ORF 3b; 5/6 = ORF 3X; 7/8 = ORF 3A Canine coronavirus 9/10 = ORF 7b; 11/12 = ORF 7a Avian infectious bronchitis virus 13/14 = ORF 5b; 15/16 = ORF 5a; 17/18 = ORF 3a; 19/20 = ORF 3b  21-520 500 primers for left part  521-1020 500 primers for right part 1021-3520 Forward primers from Table 4 3521-6020 Reverse primers from Table 4 6021-6026 FIG. 9 primers 6027-6033 FIG. 11 primers 6034-6038 Five primers from http://content.nejm.org/cgi/reprint/NEJMoa030781v2.pdf 6039-6051 PEP1 to PEP13 6052 Extended PEP13 6053-6056 229E human coronavirus sequences 6057-6060 TGV sequences 6061-6064 PEDV sequences 6065-6068 Bovine coronavirus sequences 6069-6071 Murine hepatitis virus sequences 6072-6075 AIBV sequences 6076-6170 Primer sequences (forward) 6171-6265 Primer sequences (reverse) 6266-6304 Primer sequences (forward) 6305-6343 Primer sequences (reverse) 6344-6366 Primer sequences (forward) 6367-6392 Primer sequences (reverse) 6393-6440 Primer sequences (forward) F1-F48 6441-6487 Primer sequences (reverse) R1-R47 6488-6559 Primer sequences 6560-6568 Primer sequences 6569 The nsp2 proteinase (3CL-PRO) sequence in SARS coronavirus 6570-72  The nsp2 proteinases (3CLp) of avian IBV, MHV, and BCoV 6573 Consensus nsp2 proteinases sequence 6574-6577 IG sequences from FIG. 18 6578 Expression construct of nSh in pCMVIII 6579 Expression construct of nS in pCMVIII 6580 Expression construct of nSh ΔTC in pCMVIII 6581 Expression construct of nS ΔTC in pCMVIII 6582 Expression construct of nS1h in pCMVIII 6583 Expression construct of nS1 in pCMVIII 6584-6585 Primers for cDNA amplification 6585-6587 Primers for RT-PCR 6588-6809 Component sequences of FIG. 23 (≧4 amino acids) 6810-7179 Component sequences of FIG. 24 (≧4 amino acids) 7180-7187 N-glycosylation sites within SEQ ID NO: 6039 7188-7189 Component sequences of FIG. 25 7190 Fragment of SEQ ID NO: 7188 7191 Polynucleotide encoding SEQ ID NO: 7190 7192 Amino acids 879-1005 of SEQ ID NO: 6042 7193 Amino acids 879-980 of SEQ ID NO: 6042 7194 Amino acids 901-1005 of SEQ ID NO: 6042 7195 Amino acids 1144-1201 of SEQ ID NO: 6042 7196 Amino acids 1144-1196 of SEQ ID NO: 6042 7197-7199 Membrane fusion peptide regions 7200-7206 NadA-based polypeptides 7207-7223 N-glycosylation sites within SEQ ID NO: 6042 7224-7231 Slippage region 7232 Orflab polyprotein 7233-7244 Orflab polyproteins 7245-7247 X₂ sequences for SEQ ID NOS 7233-7244 7248-7253 Orflab polyproteins 7254 Zinc binding region 2 site 7255-7271 N-glycosylation sites in SEQ ID NOS: 6040-41, 6043, 6045-46, 6050-51 7272-7291 Polypeptides and polynucleotides 7292-7293 Intergenic sequences 7294-7301 Nucleotides from 5′ end of SARSV genome followed by intergenic sequence 7302-7306 NadA constructs 7307-7308 Fragments of SEQ ID NO: 6042 7309 NadA sequence 7310-7311 NadA leader sequences 7312-7315 Amino acid sequencess from NadA 7316-7324 PCR primers 7325-7330 Primers 7331 CCACC sequence 7332-7336 3′ UTR forward primers 7337-7341 3′ UTR reverse primers 7342-7352 3′ UTR probes 7353-7362 5′ UTR forward primers 7363-7373 5′ UTR reverse primers 7374-7385 5′ UTR probes 7386 Conserved octanucleotide 7387 Reverse complement of SEQ ID NO: 7293 7388 Intergenic sequence 7389 Poly T 7390 Stem-loop sequence 7391-7392 Poly-glycine linkers 7393 Poly-histidine tag 7394 Nucleocapsid epitope site 7395 Antisense primer 7396-7397 Probes 7398-7399 Antigenic fragments of SEQ ID NO: 6042 7400-7639 T-epitope analysis of SEQ ID NO: 6039 7640-7800 T-epitope analysis of SEQ ID NO: 6040 7801-8040 T-epitope analysis of SEQ ID NO: 6041 8041-8280 T-epitope analysis of SEQ ID NO: 6042 8281-8486 T-epitope analysis of SEQ ID NO: 6043 8487-8665 T-epitope analysis of SEQ ID NO: 6044 8666-8820 T-epitope analysis of SEQ ID NO: 6045 8821-9018 T-epitope analysis of SEQ ID NO: 6046 9019-9131 T-epitope analysis of SEQ ID NO: 6047 9132-9308 T-epitope analysis of SEQ ID NO: 6048 9309-9437 T-epitope analysis of SEQ ID NO: 6049 9438-9538 T-epitope analysis of SEQ ID NO: 6050 9539-9752 T-epitope analysis of SEQ ID NO: 6052 9753-9763 Primers for spike protein amplification, particularly fragments of spike 9764-9765 N-glycosylation sites within SEQ ID NO: 6039 9766-9779 Cleavage products for ORFlab (Table 10) 9780-9782 Forward primer, reverse primer, probe 9783-9784 Lysine-rich region 9785-9798 Oligonucleotides used for S. cerevisiae expression 9799-9802 Sequences from FIGS. 65 & 66 9803-9882 Primers for E. coli cloning 9883-9885 BCV nucleotide sequences for FIGS. 3A, 3B, 3C 9886-9891 BCV amino acid sequences for FIGS. 4A, 4B, 4C, 4D, 4E, 4F 9892 BCV 5′ UTR 9893 BCV 3′ UTR 9894-9896 MHV nucleotide sequences for FIGS. 3A, 3B, 3C 9897-9902 MHV amino acid sequences for FIGS. 4A, 4B, 4C, 4D, 4E, 4F 9903-9904 AIBV nucleotide sequences for FIGS. 3A, 3B 9905-9909 AIBV amino acid sequences for FIGS. 4A, 4B, 4D, 4E, 4F 9910 AIBV 5′ UTR 9911 AIBV 3′ UTR 9912-9913 HOBMPRO, HOBHEGA nucleotide sequences for FIGS. 3B, 3C 9914-9918 Human CoV amino acid sequences for FIGS. 4A, 4B, 4C, 4E, 4F 9919 HCoV-OC43 5′ UTR 9920 HCoV-OC43 3′ UTR 9921-9923 pCMVKm2 vectors 9924-9926 Codon-optimised N, M and E sequences 9927 BNI-1 9928-9959 Constituent amino acid sequences ≧4aa inferred from SEQ ID NO: 9927 9960 ORF1ab variant 9961 ORF1a variant 9962 Spike variant 9963 Membrane variant 9964 Nucleocapsid variant 9965-9966 Short ORFs 9967 FRA complete genome

TABLE 35 Com- pound # Structure Source Literature Reference Patent Number 1

Aventis Pasteur 1) Lang. J. -M.; Touraine, J. -L.; Trepo, C, et al. Lancet 1988, 2(8613): 702-5. 2

Pfizer Dong, M. K. et al. Pharmacologist 1988, 30(3): Abst 87.8. ES 8602792 3

Mitsui Chemicals Mizuno, O. et al. 4th Int Conf Immunopharmacol (May 15-19, Osaka) 1988, Abst WS6-3. EP 236929 4

Roche EP 407788 5

Fujisawa 1) Iwami, M. et al. J Antibiot 1987, 40(5): 612-22. JP 87161796 6

Novartis FR 2604177 7

Roche Bioscience US 4725622 8

Roche Bioscience US 4727069 9

Sumitomo 1) Nishikaku, F. and Koga. Y. 4th Int Conf Immunopharmacol (May 15-19, Osaka) 1988, Abst WS6-8. EP 248399 10

SSP JP 88022053 11

Roche Bioscience US 4725622 12

Taisho 1) Kameo, K. et al. Chem Pharm Bull 1988, 36(6): 2050-60. EP 164101 13

Novartis FR 2604177 14

Novartis FR 2604177 15

Novartis FR 2604177 16

Novartis FR 2604177 17

Novartis FR 2604177 18

Novartis FR 2604177 19

Novartis FR 2604177

20

ADIR AU 8811669 21

Pharmacia AU 8810908 22

Aventis Pharma EP 284461 23

Eniricerche EP 282891 24

Pharmacia AU 8810908 25

Pharmacia AU 8810908 26

Abbott Swanson, R. N. et at. 28th Intersci Conf Antimicrob Agents Chemother (Oct 23-26, Los Angeles) 1988, Abst 972. 27

Mitsubishi Pharma JP 88119425 28

Ortho-McNeil EP 292302 29

Eniricerche EP 282891 30

Aventis Pharma EP 284461 31

ADIR AU 8811669 32

ADIR AU 8811669 33

ADIR AU 8811669 34

ADIR AU 8811669 35

ADIR AU 8811669 36

ADIR AU 8811669 37

SSP JP 88022053 38

SSP JP 88022053 39

SSP JP 88022053 40

SSP JP 88022053 41

SSP JP 88022053 42

SSP JP 88022053 43

SSP JP 88022053 44

SSP JP 88022053 45

SSP JP 88022053 46

Ortho-McNeil EP 300741 47

Aventis Pharma 1) Fizames, C. et al. 15th Int Cong Chemother (July 19-24, Istanbul) 1987, Abst 605 1) GB 2053231 48

Roche Bioscience 1) WHO Drug Inform 1988, 2(4): 227. 1) EP 135376 49

ICN 1) Sharma, B. S. et at. 3rd Intersci World Conf Inflamm, Antirheum Anaig Immunomodul (March 15-18, Monte- Carlo) 1989, 9. EP 348446 50

Toho Yakuhin 1) Satoru, I. et al. Antivir Res 1988, 9(1-2): 37-46. EP 188697 51

Gifu University 1) Hasegawa, A. et al. J Carbohyd Chem 1986, 5(3): 371-85.

52

Gifu University 53

Gifu University

54

Mitsubishi Pharma EP 351435 55

Aventis Pharma AU 8824582 56

CNRS 1) Chiron, M. et al. Biochem Pharmacol 1988, 37(5): 827-36. 57

Aventis Pharma 1) Schorlemmer, H. U.; Bartlett, R. R.; Dickneite, G.; Schwab, W.; Gebert, U.; Sedlacek, H. H. AU 8824195 58

Mitsubishi Pharma JP 88119425 59

Mitsubishi Pharma JP 88119425 60

Mitsubishi Pharma JP 88119425 61

Mitsubishi Pharma JP 88119425 62

Microbial Chemistry Research Foundation EP 310238 63

Aventis Pharma AU 8824582 64

Aventis Pharma AU 8824582 65

ICN Ramasamy, K. et al. J Med Chem 1989, 32(8): 1905-9. 66

ICN 67

ICN 68

Roche AU 8931653 69

Fujisawa Shibata, T. et at. J Antibiot 1989, 42(9): 1356-61. 70

Takeda Tanida, S. et al. J Antibiot 1989, 42(11): 1619. 71

Takeda 72

Aventis Pharma AU 8824195 73

Aventis Pharma AU 8824195 74

Aventis Pharma AU 8824195 75

Aventis Pharma AU 8824195 76

Aventis Pharma AU 8824195 77

Aventis Pharma AU 8824195 78

Aventis Pharma AU 8824195 79

Aventis Pharma AU 8824195 80

Aventis Pharma AU 8824195 81

Aventis Pharma AU 8824195 82

Aventis Pharma AU 8824195 83

Aventis Pharma AU 8824195 84

Aventis Pharma AU 8824195 85

Aventis Pharma AU 8824195 86

Aventis Pharma AU 8824195 87

Aventis Pharma AU 8824195 88

Aventis Pharma AU 8824195 89

Aventis Pharma AU 8824195 90

Microbial Chemistry Research Foundation EP 310238 91

Harbor Branch Oceanographic Institution EP 331320 92

Roche 1) Herrmann. D. B. J. 17th Int Cong Chemother (June 23-28, Berlin) 1991, Abst 838. EP 352652 93

Roche AU 8931653 94

Roche AU 8931653 95

Roche AU 8931653 96

Roche AU 8931653 97

Roche AU 8931653 98

Roche AU 8931653 99

Roche AU 8931653 100

Roche AU 8931653 101

Scharper 1) Migliorati. G. et al. 7th Int Cong Immunol (July 30-Aug 5, Berlin) 1989, Abst 106-62. 102

Mitsubishi Pharma EP 351435 103

Abbott GmbH EP 354693 104

Abbott GmbH EP 354694 105

Wyeth EP 354303 106

Merck & Co. US 4866035 107

Abbott GmbH EP 354693 108

Abbott GmbH EP 354693 109

Abbott GmbH EP 354693 110

Abbott GmbH EP 354694 111

Abbott GmbH EP354694 112

Wyeth EP 354303 113

Wyeth EP 354303 114

Wyeth EP 354303 115

Wyeth EP 354303 116

Wyeth EP 354303 117

Wyeth EP 354303 118

Wyeth EP 354303 119

Wyeth EP 354303 120

Wyeth EP 354303 121

Wyeth EP 354303 122

Wyeth EP 354303 123

Wyeth EP 354303 124

Wyeth EP 354303 125

Wyeth EP 354303 126

Wyeth EP 354303 127

Wyeth EP 354303 128

Tanabe EP 372818 129

Elan 1) Eldon, M. A. et al. J Olin Pharmacol 1990, 30(4): 352-7. 130

Lipha EP 341104 131

Tanabe EP 372818 132

Tanabe EP 372818 133

Tanabe EP 372818 134

Tanabe EP 372818 135

Tanabe EP 372818 136

Tanabe EP 372818 137

Tanabe EP 372818 138

Tanabe EP 372818 139

Tanabe EP 372818 140

Bristol-Myers Squibb US 4935493 141

Takeda JP 90193940 142

Pharmacia EP 429627 143

Merck & Co. EP 393256 144

Merck & Co. EP 393256 145

Merck & Co. EP 393256 146

Merck & Co. EP 393256 147

Bristol-Myers Squibb US 4935493 148

Bristol-Myers Squibb US 4935493 149

Bristol-Myers Squibb US 4935493 150

Bristol-Myers Squibb US 4935493 151

Bristol-Myers Squibb US 4935493 152

Sclavo EP 375058 153

SmithKline Beecham AU 9059014 154

American Biotechnology WO 9102733 155

Novartis EP 417803 156

Polifarma EP 423077 157

SCRAS GB 2228937 158

Santen EP 455833 159

American Biotechnology WO 9102733 160

Pharmacia EP 429627 161

Pharmacia EP 429627 162

Pharmacia EP 429627 163

Pharmacia EP 429627 164

Pharmacia EP 429627 165

Pharmacia EP 429627 166

Pharmacia EP 429627 167

Pharmacia EP 429627 168

Novartis EP 417803 169

Abbott GmbH WO 9111448 170

Abbott GmbH WO 9112255 171

Leland Stanford Junior University US 4996221 172

Aventis Pharma EP 438813 173

SCRAS GB 2228937 174

SCRAS GB 2228937 175

SCRAS GB 2228937 176

SCRAS GB 2228937 177

SCRAS GB 2228937 178

SCRAS GB 2228937 179

Leland Stanford Junior University US 4996221 180

Leland Stanford Junior University US 4996221 181

Leland Stanford Junior University US 4996221 182

Leland Stanford Junior University US 4996221 183

Leland Stanford Junior University US 4996221 184

Bar-llan University 1) Rephaeli, A. et al. Int J Cancer 1991, 49(1): 66. EP 302349 185

Abbott GmbH WO 9112255 186

Abbott GmbH WO 9112255 187

Abbott GmbH WO 9111448 188

Abbott GmbH WO 9111448 189

Abbott GmbH WO 9111448 190

Abbott GmbH WO 9111448 191

Abbott GmbH WO 9111448 192

Abbott GmbH WO 9111448 193

Abbott GmbH WO 9111448 194

Abbott GmbH WO 9111448 195

Bashkir Medical University Dianov, V. M. et al. Khim Farm Zh SSSR 1991, 25(1): 40. 196

Microbial Chemistry Research Foundation Kumagai, H. et al. J Antibiot 1991, 44(9): 1029. 197

Allergan US 5081261 198

Santen EP 455833 199

Santen EP 455833 200

Allergan US 5081261 201

Allergan US 5081261 202

Allergan US 5081261 203

Allergan US 5081261 204

Ono 1) Satoh, M. et al. 51st Annu Meet Jpn Cancer Assoc (Sept 29-Oct 1. Osaka) 1992. Abst 1450 EP 553786 205

Nowicky 1) Hohenwarter, O.; Strutzenberger, K.; Katinger, H.; Liepins, A.; Nowicky, J. W. Drug Exp Clin Res 1992, 18(Suppl.): 1. WO 8300486 206

Tanabe US 5210075

207

Fujisawa 1) Kurimura, M. et al. Pept Chem (1991) 1992, 361. 208

University of South Florida 1) Hadden, J. W. 5th Intersci World Conf Inflamm, Antirheum, Analg, Immunomodul (April 25-28, Geneva) 1993, Abst 257. 209

Glycomed WO 9310796 210

Karolinska Institute 1) Algarra, I. et al. Int J Cancer 1993, 54: 518. 211

Tanabe US 5210075

212

Tanabe US 5210075

213

Santen EP 558287 214

Glycomed WO 9310796 215

Glycomed WO 9310796 216

Glycomed WO 9310796 217

Microbial Chemistry Research Foundation 218

LEK 1) Sersa, G. et al. Mol Biother 1992, 4: 188. EP 477912 219

Microbial Chemistry Research Foundation 1) Ueno, M. et al. J Antibiot 1993. 46(5): 719. 220

Microbial Chemistry Research Foundation 221

Lilly 1) Bartlett, MS. et al. EP 561639 33rd lntersci Conf Antimicrob Agents Chemother (Oct 17-20, New Orleans) 1993, Abst 369. 222

Santen EP 558287 223

Santen EP 558287 224

ADIR EP 572308 225

Santen JP 93239045 226

Microbial Chemistry Research Foundation Ueno, M. et al. J Antibiot 1993, 46(11): 1658. 227

ADIR EP 572308 228

Leo WO 9401398 229

Leo WO 9401398 230

Leo WO 9401398 231

Santen JP 93239045 232

Kirin Brewery 1) Monta, M. et al. J Med Chem 1995, 38(12): 2176. 1) EP 609437 233

Kirin Brewery 1) EP 609437 234

Kirin Brewery 1) EP 609437 235

Kirin Brewery EP 609437 236

Kirin Brewery EP 609437

237

Kirin Brewery EP 609437

238

3M Pharmaceuticals Lindstrom, K. J. et al. 211th ACS Natl Meet (March 24-28, New Orleans) 1996, Abst MEDI 210. US 5482936 239

Roche Bioscience Smith, D. B. et al. J Org Chem 1996, 61(6): 2236. WO 9522538 240

AstraZeneca WO 9611943 241

AstraZeneca WO 9611943 242

AstraZeneca WO 9611943 243

AstraZeneca WO 9611943 244

AstraZeneca WO 9611943 245

AstraZeneca WO 9611943 246

AstraZeneca WO 9611943 247

AstraZeneca WO 9611943 248

AstraZeneca WO 9611943 249

Peptech WO 9612739 250

Japan Tobacco JP 96113555 251

Harvard College JP 96504177 252

Shire BioChem WO 9619494 253

Japan Tobacco JP 96113555 254

Japan Tobacco JP 96113555 255

Japan Tobacco JP 96113555 256

Shire BioChem WO 9619494 257

Harvard College JP 96504177 258

Harvard College JP 96504177 259

Harvard College JP 96504177 260

Harvard College JP 96504177 261

Harvard College JP 96504177 262

Harvard College JP 96504177 263

Japan Tobacco JP 96113555 264

Japan Tobacco JP 96113555 265

Japan Tobacco JP 96113555 266

Univ. Minnesota WO 9623793 267

Univ. Minnesota WO 9623793 268

Univ. Minnesota WO 9623793 269

Univ. Minnesota WO 9623793 270

Univ. Minnesota WO 9623793 271

AstraZeneca WO 9630397 272

AstraZeneca WO 9630397 273

AstraZeneca WO 9630397 274

AstraZeneca WO 9630397 275

Kyowa Hakko WO 9633989 276

AstraZeneca WO 9630397 277

AstraZeneca WO 9630397 278

AstraZeneca WO 9630397 279

AstraZeneca WO 9630397 280

AstraZeneca WO 9630397 281

AstraZeneca WO 9630397 282

AstraZeneca WO 9630397 283

Glycomed WO 9635700 284

Kyowa Hakko WO 9633989 285

Kyowa Hakko WO 9633989 286

Kyowa Hakko WO 9633989 287

Kyowa Hakko WO 9633989 288

Aston University Kinchington, D. et al. 4th Conf Retroviruses Opportunistic Infect (Jan 22-26, Washington DC) 1997, Abst. 289

Roche 1) Fiedler-Nagy, C. et al. Agent Action 1989, 27(3-4): 313-5. EP 169571 290

NFCR 1) Forest Laboratories, Inc. Annual Report 1994. WO 9517890 291

Sumitomo Pharmaceuticals EP 248399 292

Aventis Pharma EP 248734 293

May & Baker EP 252682 294

Sumitomo Pharmaceuticals EP 248399 295

Sumitomo Pharmaceuticals EP 248399 296

Sumitomo Pharmaceuticals EP 248399 297

Sumitomo Pharmaceuticals EP 248399 298

Sumitomo Pharmaceuticals EP 248399 299

Sumitomo Pharmaceuticals EP 248399 300

Sumitomo Pharmaceuticals EP 248399 301

Sumitomo Pharmaceuticals EP 248399 302

Aventis Pharma EP 248734 303

Aventis Pharma EP 248734 304

Aventis Pharma EP 248734 305

Aventis Pharma EP 248734 306

Aventis Pharma EP 248734 307

Aventis Pharma EP 248734 308

Pfizer AU 8783281 309

Harbor Branch Found. US 4755529 310

Roche Bioscience AU 8782540 311

Roche Bioscience AU 8782540 312

Roche Bioscience AU 8782540 313

Roche Bioscience AU 8782540 314

Novartis EP 296110 315

Novartis 1) Lam, C. et al. Antimicrob Agents Chemother 1991, 35(3): 500. AU 8822785 316

Schering-Plough EP 318214 317

Novartis AU 8822785 318

Novartis AU 8822785 319

Kyorin EP 310096 320

Kyorin EP 310096 321

Kyorin EP 310096 322

Kyorin EP 310096 323

Kyorin EP 310096 324

Kyorin EP 310096 325

Santen EP 326326 326

Roche AU 8929658 327

Taisho 1) Takeshita, K. et al. Int J Immunother 1988, 4(2): 97-106. JP 79141750 328

Harbor Branch Found. 1) Burres, N.S. et al. Proc Amer Assoc Cancer Res 1989, 30: Abst 1914. EP 331320 329

Novartis EP 296110 330

Novartis EP 296110 331

Novartis EP 296110 332

Schering-Plough EP 318214 333

Leo WO 8910351 334

Scripps Clinic Res. Found. WO 8908658 335

Scripps Clinic Res. Found. WO 8908658 336

Scripps Clinic Res. Found. WO 8908658 337

Scripps Clinic Res. Found. WO 8908658 338

Scripps Clinic Res. Found. WO 8908658 339

Scripps Clinic Res. Found. WO 8908658 340

Scripps Clinic Res. Found. WO 8908658 341

Roche AU 8929658 342

Roche AU 8929658 343

Roche AU 8929658 344

Leo WO 8910351 345

Leo WO 8910351 346

Leo WO 8910351 347

Leo WO 8910351 348

Leo WO 8910351 349

Leo WO 8910351 350

Tanabe Seiyaku 1) Ueno, M. et al. Jpn J Pharmacol 1992, 58(Suppl. 1): Abst O-210. AU 8942368 351

Aventis Pharma EP 378456 352

Greenwich Pharm. AU 9047648 353

Roche EP 384349 354

Greenwich Pharm. AU 9047648 355

Greenwich Pharm. AU 9047648 356

Aventis Pharma EP 378456 357

Aventis Pharma EP 378456 358

Aventis Pharma EP 378456 359

Aventis Pharma EP 378456 360

Aventis Pharma EP 378456 361

Aventis Pharma EP 378456 362

Aventis Pharma EP 378456 363

Leo Binderup, L. et al. Biochem Pharmacol 1991, 42(8): 1569. EP 460032 364

Microbial Chemistry Research Foundation Muraoka, Y. et al. 30th Intersci Conf Antimicrob Agents Chemother (Oct 21-24, Atlanta) 1990, Abst 801 365

Kyorin AU 9057029 366

Greenwich Pharm. AU 9057691 367

Leo EP 460032 368

Leo EP 460032 369

Leo EP 460032 370

Leo EP 460032 371

Leo EP 460032 372

Leo EP 460032 373

Leo EP 460032 374

Novartis Kricek. F. et al. Immunopharmacology 1997, 36(1): 27. AU 9057875 375

Amersham Health 1) Frey, C. L. et al. 31st Intersci Conf Antimicrob Agents Chemother (Sept 29-Oct 2, Chicago) 1991, Abst 85. AU 9059014 376

Roche EP 384349 377

Roche EP 384349 378

Roche AU 9059154 379

Kyorin AU 9057029 380

Greenwich Pharm. AU 9057691 381

Greenwich Pharm. AU 9057691 382

Greenwich Pharm. AU 9057691 383

Greenwich Pharm. AU 9057691 384

Greenwich Pharm. AU 9057691 385

Greenwich Pharm. AU 9057691 386

Greenwich Pharm. AU 9057691 387

Greenwich Pharm. AU 9057691 388

Greenwich Pharm. AU 9057691 389

Greenwich Pharm. AU 9057691 390

Greenwich Pharm. AU 9057691 391

Greenwich Pharm. AU 9057691 392

Greenwich Pharm. AU 9057691 393

Amersham Health AU 9059014 394

Amersham Health AU 9059014 395

Amersham Health AU 9059014 396

Amersham Health AU 9059014 397

Amersham Health AU 9059014 398

Amersham Health AU 9059014 399

Amersham Health AU 9059014 400

Amersham Health AU 9059014 401

Amersham Health AU 9059014 402

Leo 1) Elstner, E. et al. Blood 1994, 84(6): 1960. EP 479871 403

Leo WO 9100855 404

SPA EP 421074 405

Hitachi Chemical EP 421682 406

Roche AU 9059154 407

Roche AU 9059154 408

Roche AU 9059154 409

Roche AU 9059154 410

Roche AU 9059154 411

Fujisawa 1) Manda, T. et al. Jpn J Pharmacol 1997, 73(Suppl. 1): Abst P-140. EP 412404 412

Allergan US 5013850 413

Leo WO 9100855 414

Leo WO 9100855 415

Leo WO 9100855 416

Leo WO 9100855 417

Leo EP 479871 418

Leo EP 479871 419

Leo WO 9109841 420

Hitachi Chemical EP 421682 421

Hitachi Chemical EP 421682 422

Hitachi Chemical EP 421682 423

Hitachi Chemical EP 421682 424

Hitachi Chemical EP 421682 425

Hitachi Chemical EP 421682 426

Hitachi Chemical EP 421682 427

Hitachi Chemical EP 421682 428

SPA EP 421074 429

SPA EP 421074 430

SPA EP 421074 431

SPA EP 421074 432

SPA EP 421074 433

SPA EP 421074 434

SPA EP 421074 435

SPA EP 421074 436

SPA EP 421074 437

SPA EP 421074 438

SPA EP 421074 439

SPA EP 421074 440

SPA EP 421074 441

SPA EP 421074 442

SPA EP 421074 443

SPA EP 421074 444

Allergan US 5013850 445

Allergan US 5013850 446

Allergan US 5013850 447

Allergan US 5013850 448

Allergan US 5013850 449

Allergan US 5013850 450

Allergan US 5013850 451

Allergan US 5013850 452

Allergan US 5013850 453

Allergan US 5013850 454

Allergan US 5013850 455

Allergan US 5013850 456

Allergan US 5013850 457

Allergan US 5013850 458

Allergan US 5013850 459

Allergan US 5013850 460

Allergan US 5013850 461

New England Med. Center Hosp. WO 9116339 462

Leo EP 506794 463

Fujisawa WO 9119708 464

Roche 1) Hill, C. H. 6th SCI- RSC Med Chem Symp (Sept 8-11, Cambridge) 1991, Abst S18. EP 384349 465

New England Med. Center Hosp. WO 9116339 466

Alder US 5145842 467

Harbor Branch Found. US 5091368 468

Aventis Pharma EP 476658 469

Merck & Co. EP 480713 470

Alder US 5145842 471

Alder US 5145842 472

Alder US 5145842 473

Alder US 5145842 474

Alder US 5145842 475

Alder US 5145842 476

Alder US 5145842 477

Aventis Pharma EP 476658 478

Aventis Pharma EP 476658 479

Aventis Pharma EP 476658 480

Aventis Pharma EP 476658 481

Aventis Pharma EP 476658 482

Aventis Pharma EP 476658 483

Aventis Pharma EP 476658 484

Aventis Pharma EP 476658 485

Harbor Branch Found. US 5091368 486

Merck & Co. EP 480713 487

Merck & Co. EP 480713 488

Merck & Co. EP 480713 489

Merck & Co. EP 480713 490

Kyowa Hakko Miwa, K. et al. 113th Annu Meet Pharmaceut Soc Jpn (March 29-31, Osaka) 1993, Abst 30CC 13-1. EP 505058 491

Roche EP 510473 492

Beaufour-Ipsen 1) Carde, P. et al. Proc Amer Soc Clin Oncol 1991, 10: Abst 324. AU 8810261 493

Roche EP 510473 494

Roche EP 510473 495

Roche EP 510473 496

Fujisawa WO 9218483 497

Kyowa Hakko EP 526840 498

Kyowa Hakko EP 526840 499

Kyowa Hakko EP 526840 500

Aventis Pharma EP 538783 501

Fujisawa Nakamura, K. et al. Chem Pharm Bull 1993, 41(5): 894. AU 8783152 502

Hayashibara 1) Yamamoto, I. et al. 18th Int Cong Chemother (June 27-July 2, Stockholm) 1993, Abst 516. EP 539196 503

Kyowa Hakko WO 9312116 504

GlaxoSmithKline WO 9314082 505

Otsuka JP 93132484 506

Pfizer US 5236926 507

Aventis Pharma EP 538783 508

Aventis Pharma EP 538783 509

GlaxoSmithKline WO 9314082 510

GlaxoSmithKline WO 9314082 511

GlaxoSmithKline WO 9314082 512

GlaxoSmithKline WO 9314082 513

GlaxoSmithKline WO 9314082 514

GlaxoSmithKline WO 9314082 515

Otsuka JP 93132484 516

Otsuka JP 93132484 517

Otsuka JP 93132484 518

Otsuka JP 93132484 519

Otsuka JP 93132484 520

Pfizer US 5236926 521

Pfizer US 5236926 522

Pfizer US 5236926 523

Pfizer US 5236926 524

Pfizer US 5236926 525

Sumitomo US 5258396 526

Wyeth US 5312831 527

Cell Therapeutics WO 9411001 528

Greenwich Pharm. WO 9411381 529

Otsuka JP 94100561 530

Wyeth US 5312831 531

Wyeth US 5312831 532

Cell Therapeutics WO 9411001 533

Cell Therapeutics WO 9411001 534

Cell Therapeutics WO 9411001 535

Cell Therapeutics WO 9411001 536

Cell Therapeutics WO 9411001 537

Cell Therapeutics WO 9411001 538

Cell Therapeutics WO 9411001 539

Cell Therapeutics WO 9416704 540

Immunex 1) Immunex Corporation Press Release 1994, July 21. WO 9506031 541

Greenwich Pharm. WO 9411381 542

Greenwich Pharm. WO 9411381 543

Greenwich Pharm. WO 9411381 544

Greenwich Pharm. WO 9411381 545

Greenwich Pharm. WO 9411381 546

Greenwich Pharm. WO 9411381 547

Greenwich Pharm. WO 9411381 548

Greenwich Pharm. WO 9411381 549

Greenwich Pharm. WO 9411381 550

Greenwich Pharm. WO 9411381 551

Greenwich Pharm. WO 9411381 552

Greenwich Pharm. WO 9411381 553

British Technol. GB 2278842 554

Cell Therapeutics WO 9416704 555

Cell Therapeutics WO 9416704 556

Cell Therapeutics WO 9416704 557

Cell Therapeutics WO 9416704 558

Cell Therapeutics WO 9416704 559

Cell Therapeutics WO 9416704 560

Cell Therapeutics WO 9416704 561

Otsuka JP 94100561 562

Otsuka JP 94100561 563

Otsuka JP 94100561 564

Otsuka JP 94100561 565

Otsuka JP 94100561 566

Otsuka JP 94100561 567

Otsuka JP 94100561 568

Otsuka JP 94100561 569

Otsuka JP 94100561 570

Otsuka JP 94100561 571

Abbott GmbH WO 9500493 572

Abbott GmbH WO 9500507 573

Leo WO 9502577 574

GlaxoSmithKline WO 9504734 575

Kyorin Kono, Y. et al. 115th Annu Meet Pharmaceut Soc Jpn (March 29-31, Sendai) 1995, Abst 30 (A3) 10-3. EP 538477 576

Abbott GmbH WO 9500507 577

Abbott GmbH WO 9500507 578

Abbott GmbH WO 9500507 579

Abbott GmbH WO 9500493 580

Abbott GmbH WO 9500493 581

Abbott GmbH WO 9500493 582

Abbott GmbH WO 9500493 583

Abbott GmbH WO 9500493 584

Abbott GmbH WO 9500493 585

Abbott GmbH WO 9500493 586

Abbott GmbH WO 9500493 587

Abbott GmbH WO 9500493 588

Abbott GmbH WO 9500493 589

Abbott GmbH WO 9500493 590

Sanofi- Synthelabo EP 644197 591

Sanofi- Synthelabo CA 2125021 592

Kyowa Hakko WO 9509153 593

GlaxoSmithKline WO 9504734 594

GlaxoSmithKline WO 9504734 595

GlaxoSmithKline WO 9504734 596

GlaxoSmithKline WO 9504734 597

GlaxoSmithKline WO 9504734 598

GlaxoSmithKline WO 9504734 599

Japan Tobacco JP 95002779 600

Pharmacia 1) Gozzi, P. et al. J Pharmacol Exp Ther 1999, 291(1): 199. JP 1995501330 601

Bayer US 5409932 602

Bayer US 5411960 603

Sanofi- Synthelabo EP 644197 604

Sanofi- Synthelabo EP 644197 605

Sanofi- Synthelabo EP 644197 606

Sanofi- Synthelabo CA 2125021 607

Sanofi- Synthelabo CA 2125021 608

Sanofi- Synthelabo CA 2125021 609

Sanofi- Synthelabo CA 2125021 610

Sanofi- Synthelabo CA 2125021 611

Sanofi- Synthelabo CA 2125021 612

Sanofi- Synthelabo CA 2125021 613

Baxter France, C. P. et al. Drug Develop Res 1995, 35: 49. EP 396282 614

Bayer U5 5411960 615

Bayer US 5411960 616

Bayer US 5411960 617

Bayer US 5411960 618

Bayer US 5409932 619

Lilly WO 9517382 620

Millennium WO 9518610 621

Aventis Pharma WO 9520578 622

Cell Therapeutics WO 9522546 623

Lilly WO 9517382 624

Lilly WO 9517382 625

Lilly WO 9517382 626

Lilly WO 9517382 627

Lilly WO 9517382 628

Lilly WO 9517382 629

Lilly WO 9517382 630

Lilly WO 9517382 631

Lilly WO 9517382 632

CytoMed WO 9518610 633

CytoMed WO 9518610 634

CytoMed WO 9518610 635

CytoMed WO 9518610 636

CytoMed WO 9518610 637

CytoMed WO 9518610 638

CytoMed WO 9518610 639

CytoMed WO 9518610 640

CytoMed WO 9518610 641

CytoMed WO 9518610 642

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CytoMed WO 9518610 644

CytoMed WO 9518610 645

CytoMed WO 9518610 646

CytoMed WO 9518610 647

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CytoMed WO 9518610 650

Duphar EP 664287 651

Japan Tobacco JP 95002779 652

Japan Tobacco JP 95002779 653

Japan Tobacco JP 95002779 654

Japan Tobacco JP 95002779 655

Japan Tobacco JP 95002779 656

Japan Tobacco JP 95002779 657

Japan Tobacco JP 95002779 658

Japan Tobacco JP 95002779 659

Aventis Pharma WO 9520578 660

Aventis Pharma WO 9520578 661

Aventis Pharma WO 9520578 662

Aventis Pharma WO 9520578 663

Kyowa Hakko WO 9509153 664

Kyowa Hakko WO 9509153 665

Kyowa Hakko WO 9509153 666

Kyowa Hakko WO 9509153 667

Kyowa Hakko WO 9509153 668

Kyowa Hakko WO 9509153 669

Kyowa Hakko WO 9509153 670

Sanofi- Synthelabo WO 9526958 671

Cell Therapeutics WO 9522546 672

Cell Therapeutics WO 9522546 673

Cell Therapeutics WO 9522546 674

Cell Therapeutics WO 9522546 675

Cell Therapeutics WO 9522546 676

Cell Therapeutics WO 9522546 677

Duphar EP 664287 678

Duphar EP 664287 679

Duphar EP 664287 680

Duphar EP 664287 681

Duphar EP 664287 682

Duphar EP 664287 683

Sanofi- Synthelabo WO 9529672 684

Sanofi- Synthelabo WO 9526958 685

Sanofi- Synthelabo WO 9526958 686

Takeda WO 9535296 687

Vertex WO 9535308 688

Daikin EP 711766 689

Sanofi- Synthelabo WO 9533751 690

Entropin WO 9534561 691

Takeda WO 9535296 692

Takeda WO 9535296 693

Takeda WO 9535296 694

Takeda WO 9535296 695

Vertex WO 9535308 696

Vertex WO 9535308 697

Vertex WO 9535308 698

Ajinomoto US 5464819 699

Sanofi- Synthelabo WO 9529672 700

Sanofi- Synthelabo WO 9529672 701

Sanofi- Synthelabo WO 9529672 702

3M Pharmaceuticals US 5482936 703

3M Pharmaceuticals US 5482936 704

3M Pharmaceuticals US 5482936 705

3M Pharmaceuticals US 5482936 706

Entropin WO 9534561 707

Entropin WO 9534561 708

Entropin WO 9534561 709

Entropin WO 9534561 710

Entropin WO 9534561 711

Entropin WO 9534561 712

Entropin WO 9534561 713

Ajinomoto US 5464819 714

Ajinomoto US 5464819 715

Ajinomoto US 5464819 716

Sanofi- Synthelabo WO 9533751 717

Sanofi- Synthelabo WO 9533751 718

Sanofi- Synthelabo WO 9533751 719

Sanofi- Synthelabo WO 9533751 720

Sanofi- Synthelabo WO 9533751 721

Sanofi- Synthelabo WO 9533751 722

Daikin EP 711766 723

Daikin EP 711766 724

Daikin EP 711766 725

Daikin EP 711766 726

Taisho 1) Yoshida, H. et al. Biol Pharm Bull 1997, 20(1): 94. JP 93051358 727

Microbial Chemistry Research Foundation JP 96176157

 728

Sankyo Shiozaki, M. et al. Tetrahedron Lett 1996 37(40): 7271.  729

Nippon Kayaku JP 96283290  730

Tanabe WO 9640641  731

Tanabe WO 9640641  732

Daiichi Pharma- ceutical 1) Kawagoe, K. et al. AFMC Int Med Chem Symp (Sept 3-8, Tokyo) 1995, Abst P13M183. JP 97059236  733

Vertex WO 9722618  734

Vertex WO 9722618  735

Vertex WO 9722618  736

Vertex WO 9722618  737

Vertex WO 9722618  738

Vertex WO 9722618  739

Astra Zeneca WO 9731023  740

Univer- sity of Penn- sylvania WO 9733603  741

Eisai EP 889032  742

AWD. pharma WO 9734874  743

Astra Zeneca WO 9731023  744

Astra Zeneca WO 9731023  745

Astra Zeneca WO 9731023  746

Astra Zeneca WO 9731023  747

AWD. pharma WO 9734874  748

AWD. pharma WO 9734874  749

Leo WO 9737972  750

Taisho JP 97194476  751

Leo WO 9737972  752

Leo WO 9737972  753

Leo WO 9737972  754

Leo WO 9737972  755

Leo WO 9737972  756

Glaxo- Smith Kline WO 9743250  757

Astra Zeneca 1) Saem- strand, B. et al. J Pharmacol Exp Ther 1999, 228(3): 1174. EP 0463514  758

Pharm- acia WO 9745409  759

Pharm- acia WO 9745409  760

Pharm- acia WO 9745409  761

Pharm- acia WO 9745409  762

Eisai Nagai, M. et al. 217th ACS Natl Meet (March 21-25, Ana- heim) 1999, Abst MEDI 050. EP 889032  763

Bashkir Medical Univer- sity Sadykov, R. F. et al. Naunyn- Schmied Arch Pharm- acol 1998, 358(1, Suppl. 2): Abst P 52.28.  764

LEK EP 477912  765

Gruen- enthal EP 856513  766

Gruen- enthal EP 856513  767

Gruen- enthal EP 856513  768

Gruen- enthal EP 856513  769

Astra Zeneca WO 9828275  770

Astra Zeneca WO 9828275  771

Astra Zeneca WO 9828270  772

Astra Zeneca WO 9828270  773

Astra Zeneca WO 9828270  774

Astra Zeneca WO 9828270  775

Astra Zeneca WO 9828270  776

Astra Zeneca WO 9828270  777

Astra Zeneca WO 9828270  778

Astra Zeneca WO 9828270  779

Astra Zeneca WO 9828270  780

Astra Zeneca WO 9828270  781

Astra Zeneca WO 9828270  782

Astra Zeneca WO 9828270  783

Astra Zeneca WO 9828270  784

Astra Zeneca WO 9828270  785

Astra Zeneca WO 9828270  786

Astra Zeneca WO 9828270  787

Astra Zeneca WO 9828270  788

Astra Zeneca WO 9828270  789

Japan Energy JP 98231297  790

Japan Energy JP 98231297  791

Japan Energy JP 98231297  792

Japan Energy JP 98231297  793

Japan Energy JP 98231297  794

Celgene 1) Moreira, A. L. et al. J Neuro- Oncol 1999, 43(2): 109. U.S. Pat. No. 5463063  795

Daiichi Pharma- ceutical Koiwa, T. et al. J Antibiot 1999, 52(2): 198.  796

Celgene U.S. Pat. No. 5874448  797

Celgene U.S. Pat. No. 5874448  798

Celgene U.S. Pat. No. 5874448  799

Celgene U.S. Pat. No. 5874448  800

Celgene U.S. Pat. No. 5874448  801

Eisai EP 889032  802

Merck & Co. WO 9909984  803

Merck & Co. WO 9909984  804

Merck & Co. WO 9909984  805

Merck & Co. WO 9909984  806

Hokur- iku JP 99080156  807

Hokur- iku JP 99080156  808

Hokur- iku JP 99080156  809

Hokur- iku JP 99080156  810

Hokur- iku JP 99080156  811

SSP CA 2255337  812

SSP CA 2255337  813

SSP CA 2255337  814

Abbott Madar, D. et al. 222nd ACS Natl Meet (Aug 26-30, Chicago) 2001, Abst MEDI 7. EP 1068187  815

Active Biotech WO 9955678  816

Amer- sham 1) Gesser, B. et al. Proc Natl Acad Sci USA 1997, 94(26): 14620. WO 9601318  817

Kowa WO 9944995  818

Kowa WO 9944995  819

Kowa WO 9944995  820

OM Pharma WO 0000462  821

OM Pharma WO 0000462  822

Ono WO 0003980  823

Ono WO 0003980  824

Ono WO 0003980  825

Ono WO 0003980  826

Ono WO 0003980  827

Ono WO 0003980  828

Ono WO 0003980  829

Univer- sity of Bristol WO 0014114  830

Eisai Hibi, S. et al. Bioorg Med Chem Lett 2000, 10(7): 623. EP 0889032  831

Janssen WO 0021959  832

Janssen WO 0021959  833

Janssen WO 0021959  834

Janssen WO 0021959  835

Fuji- sawa WO 0021979  836

Abbott 1) Liu, G. et al. 220th ACS Natl Meet (Aug 20-24, Washington DC) 2000, Abst MEDI 171. 1) U.S. Pat. No. 6110922  837

Cancer Re- search UK WO 0052046  838

Cancer Re- search UK WO 0052046  839

Cancer Re- search UK WO 0052046  840

South- ern Re- search Institute WO 0112197  841

South- ern Re- search Institute WO 0112197  842

Abbott Labs. EP 1068187  843

Abbott EP 1068187  844

Abbott EP 1068187  845

Fuji- sawa Spears, G. et al. 21st Symp Med Chem (Nov 28-30, Kyoto) 2001, Abst 2P-29.  846

Austin Re- search Institute Tselios, T. et al. J Med Chem 2002, 45(2): 275.  847

Eukar- ion WO 0204454  848

Nobex 1) Riggs- Sauthier, J. A. et al. 224th ACS Natl Meet (Aug 18-22, Boston) 2002, Abst MEDI 305. WO 0218324  849

Tularik WO 0238107  850

Tularik WO 0238107  851

Tularik WO 0238107  852

Tularik WO 0238107  853

Tularik WO 0238107  854

Tularik WO 0238107  855

Tularik WO 0238107  856

Tularik WO 0238107  857

Tularik WO 0238107  858

Sanofi- Synthe labo WO 0242269  859

Sanofi- Synthe labo WO 0242269  860

Sanofi- Synthe labo WO 0242269  861

Sanofi- Synthe labo WO 0242269  862

Sanofi- Synthe labo WO 0242269  863

Sanofi- Synthe labo WO 0242269  864

Sanofi- Synthe labo WO 0242269  865

Sanofi- Synthe labo WO 0242269  866

Sanofi- Synthe labo WO 0242269  867

Bristol- Myers Squibb WO 0244181  868

Bristol- Myers Squibb WO 0244181  869

Bristol- Myers Squibb WO 0244181  870

Bristol- Myers Squibb WO 0244181  871

Bristol- Myers Squibb WO 0244181  872

Bristol- Myers Squibb WO 0244181  873

Bristol- Myers Squibb WO 0244181  874

Bristol- Myers Squibb WO 0244181  875

Bristol- Myers Squibb WO 0244181  876

Cell Thera- peutics WO 0268421  877

Cell Thera- peutics WO 0268421  878

Gruen- enthal WO 0290317  881

May & Baker EP 252682  882

Kyorin EP 310096  883

Kyorin EP 310096  884

Kyorin EP 310096  885

Kyorin EP 310096  886

Kyorin EP 310096  887

Kyorin EP 310096  888

Santen EP 326326  889

Roche AU 8929658  890

Taisho 1) Takeshita, K. et al. Int J Immunother 1988, 4(2): 97-106. JP 79141750  891

Harbor Branch Found. 1) Burres, N. S. et al. Proc Amer Assoc Cancer Res 1989, 30: Abst 1914. EP 331320  892

Scripps Clinic Res. Found. WO 8908658  893

Scripps Clinic Res. Found. WO 8908658  894

Scripps Clinic Res. Found. WO 8908658  895

Scripps Clinic Res. Found. WO 8908658  896

Scripps Clinic Res. Found. WO 8908658  897

Scripps Clinic Res. Found. WO 8908658  898

Scripps Clinic Res. Found. WO 8908658  899

Roche AU 8929658  900

Roche AU 8929658  901

Roche AU 8929658  902

Aventis Pharma EP 378456  903

Roche EP 384349  904

Green- wich Pharm. AU 9047648  905

Aventis Pharma EP 378456  906

Aventis Pharma EP 378456  907

Aventis Pharma EP 378456  908

Aventis Pharma EP 378456  909

Aventis Pharma EP 378456  910

Leo Binderup, L. et al. Biochem Pharmacol 1991, 42(8): 1569. EP 460032  911

Micro- bial Chem- istry Re- search Founda- tion Muraoka, Y. et al. 30th Intersci Conf Antimicrob Agents Chemother (Oct 21-24, Atlanta) 1990, Abst 801.  912

Kyorin AU 9057029  913

Green- wich Pharm. AU 9057691  914

Leo EP 460032  915

Leo EP 460032  916

Leo EP 460032  917

Leo EP 460032  918

Leo EP 460032  919

Leo EP 460032  920

Leo EP 460032  921

Novar- tis Kricek, F. et al. Immuno- pharmacol- ogy 1997, 36(1): 27. AU 9057875  922

Amer- sham Health 1) Frey, C. L. et al. 31st Intersci Conf Anti- microb Agents Chemother (Sept 29-Oct 2, Chicago) 1991, Abst 85. AU 9059014  923

Roche EP 384349  924

Roche AU 9059154  925

Kyorin AU 9057029  926

Green- wich Pharm. AU 9057691  927

Green- wich Pharm. AU 9057691  928

Green- wich Pharm. AU 9057691  929

Green- wich Pharm. AU 9057691  930

Green- wich Pharm. AU 9057691  931

Green- wich Pharm. AU 9057691  932

Green- wich Pharm. AU 9057691  933

Green- wich Pharm. AU 9057691  934

Green- wich Pharm. AU 9057691  935

Green- wich Pharm. AU 9057691  936

Green- wich Pharm. AU 9057691  937

Green- wich Pharm. AU 9057691  938

Green- wich Pharm. AU 9057691  939

Amer- sham Health AU 9059014  940

Amer- sham Health AU 9059014  941

Amer- sham Health AU 9059014  942

Amer- sham Health AU 9059014  943

Amer- sham Health AU 9059014  944

Amer- sham Health AU 9059014  945

Amer- sham Health AU 9059014  946

Amer- sham Health AU 9059014  947

Amer- sham Health AU 9059014  948

Leo 1) Elstner, E. et al. Blood 1994, 84(6): 1960. 1) EP 479871  949

Leo WO 9100855  950

Roche AU 9059154  951

Roche AU 9059154  952

Roche AU 9059154  953

Roche AU 9059154  954

Roche AU 9059154  955

Aller- gan U.S. Pat. No. 5013850  956

Leo WO 9100855  957

Leo WO 9100855  958

Leo WO 9100855  959

Leo WO 9100855  960

Leo EP 479871  961

Leo EP 479871  962

Aller- gan U.S. Pat. No. 5013850  963

Aller- gan U.S. Pat. No. 5013850  964

Aller- gan U.S. Pat. No. 5013850  965

Aller- gan U.S. Pat. No. 5013850  966

Aller- gan U.S. Pat. No. 5013850  967

Aller- gan U.S. Pat. No. 5013850  968

Aller- gan U.S. Pat. No. 5013850  969

Aller- gan U.S. Pat. No. 5013850  970

Aller- gan U.S. Pat. No. 5013850  971

Aller- gan U.S. Pat. No. 5013850  972

Aller- gan U.S. Pat. No. 5013850  973

Aller- gan U.S. Pat. No. 5013850  974

Aller- gan U.S. Pat. No. 5013850  975

Aller- gan U.S. Pat. No. 5013850  976

Aller- gan U.S. Pat. No. 5013850  977

Aller- gan U.S. Pat. No. 5013850  978

Aller- gan U.S. Pat. No. 5013850  979

New England Med. Center Hosp. WO 9116339  980

Roche 1) Hill, C. H. 6th SCI-RSC Med Chem Symp (Sept 8-11, Cam- bridge) 1991, Abst S18. EP 384349  981

New England Med. Center Hosp. WO 9116339  982

Adler U.S. Pat. No. 5145842  983

Adler U.S. Pat. No. 5145842  984

Adler U.S. Pat. No. 5145842  985

Adler U.S. Pat. No. 5145842  986

Adler U.S. Pat. No. 5145842  987

Adler U.S. Pat. No. 5145842  988

Adler U.S. Pat. No. 5145842  989

Adler U.S. Pat. No. 5145842  990

Roche EP 510473  991

Beau- four- Ipsen 1) Carde, P. et al. Proc Amer Soc Clin Oncol 1991, 10: Abst 324. AU 8810261  992

Roche EP 510473  993

Roche EP 510473  994

Roche EP 510473  995

Fuji- sawa WO 9218483  996

Kyowa Hakko EP 526840  997

Kyowa Hakko EP 526840  998

Kyowa Hakko EP 526840  999

Aventis Pharma EP 538783 1000

Haya- shibara 1) Yama- moto, I. et al. 18th Int Cong Chemother (June 27-July 2, Stockholm) 1993, Abst 516. EP 539196 1001

Kyowa Hakko WO 9312116 1002

Glaxo Smith Kline WO 9314082 1003

Otsuka JP 93132484 1004

Pfizer U.S. Pat. No. 5236926 1005

Aventis Pharma EP 538783 1006

Aventis Pharma EP 538783 1007

Glaxo Smith Kline WO 9314082 1008

Glaxo Smith Kline WO 9314082 1009

Glaxo Smith Kline WO 9314082 1010

Glaxo Smith Kline WO 9314082 1011

Glaxo Smith Kline WO 9314082 1012

Glaxo Smith Kline WO 9314082 1013

Otsuka JP 93132484 1014

Otsuka JP 93132484 1015

Otsuka JP 93132484 1016

Otsuka JP 93132484 1017

Otsuka JP 93132484 1018

Pfizer U.S. Pat. No. 5236926 1019

Pfizer U.S. Pat. No. 5236926 1020

Pfizer U.S. Pat. No. 5236926 1021

Pfizer U.S. Pat. No. 5236926 1022

Pfizer U.S. Pat. No. 5236926 1023

Sumi- tomo U.S. Pat. No. 5258396 1024

Cell Thera- peutics WO 9411001 1025

Green- wich Pharm. WO 9411381 1026

Otsuka JP 94100561 1027

Cell Thera- peutics WO 9411001 1028

Cell Thera- peutics WO 9411001 1029

Cell Thera- peutics WO 9411001 1030

Cell Thera- peutics WO 9411001 1031

Cell Thera- peutics WO 9411001 1032

Cell Thera- peutics WO 9411001 1033

Cell Thera- peutics WO 9411001 1034

Green- wich Pharm. WO 9411381 1035

Green- wich Pharm. WO 9411381 1036

Green- wich Pharm. WO 9411381 1037

Green- wich Pharm. WO 9411381 1038

Green- wich Pharm. WO 9411381 1039

Green- wich Pharm. WO 9411381 1040

Green- wich Pharm. WO 9411381 1041

Green- wich Pharm. WO 9411381 1042

Green- wich Pharm. WO 9411381 1043

Green- wich Pharm. WO 9411381 1044

Green- wich Pharm. WO 9411381 1045

Green- wich Pharm. WO 9411381 1046

Bristol Tech- nol. GB 2278842 1047

Otsuka JP 94100561 1048

Otsuka JP 94100561 1049

Otsuka JP 94100561 1050

Otsuka JP 94100561 1051

Otsuka JP 94100561 1052

Otsuka JP 94100561 1053

Otsuka JP 94100561 1054

Otsuka JP 94100561 1055

Otsuka JP 94100561 1056

Otsuka JP 94100561 1057

Abbott GmbH WO 9500507 1058

Leo WO 9502577 1059

Kyorin Kono, Y. et al. 115th Annu Meet Pharmaceut Soc Jpn March 29-31, Sendai) 1995, Abst 30 (A3) 10-3. EP 538477 1060

Abbott GmbH WO 9500507 1061

Abbott GmbH WO 9500507 1062

Abbott GmbH WO 9500507 1063

Kyowa Hakko WO 9509153 1064

Bayer U.S. Pat. No. 5409932 1065

Bayer U.S. Pat. No. 5411960 1066

Baxter France, C. P. et al. Drug Develop Res 1995, 35:49. EP 396282 1067

Bayer U.S. Pat. No. 5411960 1068

Bayer U.S. Pat. No. 5411960 1069

Bayer U.S. Pat. No. 5411960 1070

Bayer U.S. Pat. No. 5411960 1071

Bayer U.S. Pat. No. 5409932 1072

Kyowa Hakko WO 9509153 1073

Kyowa Hakko WO 9509153 1074

Kyowa Hakko WO 9509153 1075

Kyowa Hakko WO 9509153 1076

Kyowa Hakko WO 9509153 1077

Kyowa Hakko WO 9509153 1078

Kyowa Hakko WO 9509153 1079

Sanofi- Synthe- labo WO 9529672 1080

Takeda WO 9535296 1081

Vertex WO 9535308 1082

Entro pin WO 9534561 1083

Takeda WO 9535296 1084

Takeda WO 9535296 1085

Takeda WO 9535296 1086

Takeda WO 9535296 1087

Vertex WO 9535308 1088

Vertex WO 9535308 1089

Vertex WO 9535308 1090

Sanofi- Synthe- labo WO 9529672 1091

Sanofi- Synthe- labo WO 9529672 1092

Sanofi- Synthe- labo WO 9529672 1093

3M Pharma- ceuti cals U.S. Pat. No. 5482936 1094

3M Pharma- ceuti cals U.S. Pat. No. 5482936 1095

3M Pharma- ceuti cals U.S. Pat. No. 5482936 1096

3M Pharma- ceuti cals U.S. Pat. No. 5482936 1097

Entro- pin WO 9534561 1098

Entro- pin WO 9534561 1099

Entro- pin WO 9534561 1100

Entro- pin WO 9534561 1101

Entro- pin WO 9534561 1102

Entro- pin WO 9534561 1103

Entro- pin WO 9534561 1104

Taisho 1) Yoshida, H. et al. Biol Pharm Bull 1997, 20(1): 94. JP 93051358 1105

Sankyo Shiozaki, M. et al. Tetrahedron Lett 1996 37(40): 7271. 1106

Nippon Kayaku JP 96283290 1107

Eisai EP 889032 1108

AWD. pharma WO 9734874 1109

AWD. pharma WO 9734874 1110

AWD. pharma WO 9734874 1111

Astra Zeneca 1) Saem- strand, B. et al. J Pharm- acol Exp Ther 1999, 228(3): 1174. EP 0463514 1112

Eisai Nagai, M. et al. 217th ACS Natl Meet (March 21-25, Ana- heim) 1999, Abst MEDI 050. EP 889032 1113

Bashkir Medical Univer- sity Sadykov, R. F. et al. Naunyn- Schmied Arch Pharm- acol 1998, 358(1, Suppl. 2): Abst P 52.28. 1114

Gruen- enthal EP 856513 1115

Gruen- enthal EP 856513 1116

Gruen- enthal EP 856513 1117

Gruen- enthal EP 856513 1118

Japan Energy JP 98231297 1119

Japan Energy JP 98231297 1120

Japan Energy JP 98231297 1121

Japan Energy JP 98231297 1122

Japan Energy JP 98231297 1123

Celgene 1) Moreira, A. L. et al. J Neuro- Oncol 1999, 43(2): 109. U.S. Pat. No. 5463063 1124

Celgene U.S. Pat. No. 5874448 1125

Celgene U.S. Pat. No. 5874448 1126

Celgene U.S. Pat. No. 5874448 1127

Celgene U.S. Pat. No. 5874448 1128

Celgene U.S. Pat. No. 5874448 1129

Eisai EP 889032 1130

Merck & Co. WO 9909984 1131

Merck & Co. WO 9909984 1132

Merck & Co. WO 9909984 1133

Merck & Co. WO 9909984 1134

Hokur- iku JP 99080156 1135

Hokur- iku JP 99080156 1136

Hokur- iku JP 99080156 1137

Hokur- iku JP 99080156 1138

Hokur- iku JP 99080156 1139

SSP CA 2255337 1140

SSP CA 2255337 1141

SSP CA 2255337 1142

Abbott Madar, D. et al. 222nd ACS Natl Meet (Aug 26-30, Chicago) 2001, Abst MEDI 7. EP 1068187 1143

Active Biotech WO 9955678 1144

Amer- sham 1) Gesser, B. et al. Proc Natl Acad Sci USA 1997, 94(26): 14620. WO 9601318 1145

Kowa WO 9944995 1146

Kowa WO 9944995 1147

Kowa WO 9944995 1148

OM Pharma WO 0000462 1149

OM Pharma WO 0000462 1150

Ono WO 0003980 1151

Ono WO 0003980 1152

Ono WO 0003980 1153

Ono WO 0003980 1154

Ono WO 0003980 1155

Ono WO 0003980 1156

Ono WO 0003980 1157

Univer- sity of Bristol WO 0014114 1158

Eisai Hibi, S. et al. Bioorg Med Chem Lett 2000, 10(7): 623. EP 0889032 1159

Janssen WO 0021959 1160

Janssen WO 0021959 1161

Janssen WO 0021959 1162

Janssen WO 0021959 1163

Fuji- sawa WO 0021979 1164

Abbott 1) Liu, G. et al. 220th ACS Natl Meet (Aug 20-24, Washington DC) 2000, Abst MEDI 171. 1) U.S. Pat. No. 6110922 1165

Cancer Re- search UK WO 0052046 1166

Cancer Re- search UK WO 0052046 1167

Cancer Re- search UK WO 0052046 1168

South- ern Re- search Institute WO 0112197 1169

South- ern Re- search Institute WO 0112197 1170

Abbott Labs. EP 1068187 1171

Abbott EP 1068187 1172

Abbott EP 1068187 1173

Fuji- sawa Spears, G. et al. 21st Symp Med Chem (Nov 28-30, Kyoto) 2001, Abst 2P-29. 1174

Austin Re- search Institute Tselios, T. et al. J Med Chem 2002, 45(2): 275. 1175

Eukar- ion WO 0204454 1176

Nobex 1) Riggs- Sauthier, J. A. et al. 224th ACS Natl Meet (Aug 18-22, Boston) 2002, Abst MEDI 305. WO 0218324 1177

Tularik WO 0238107 1178

Tularik WO 0238107 1179

Tularik WO 0238107 1180

Tularik WO 0238107 1181

Tularik WO 0238107 1182

Tularik WO 0238107 1183

Tularik WO 0238107 1184

Tularik WO 0238107 1185

Tularik WO 0238107 1186

Sanofi- Synthe labo WO 0242269 1187

Sanofi- Synthe labo WO 0242269 1188

Sanofi- Synthe labo WO 0242269 1189

Sanofi- Synthe labo WO 0242269 1190

Sanofi- Synthe labo WO 0242269 1191

Sanofi- Synthe labo WO 0242269 1192

Sanofi- Synthe labo WO 0242269 1193

Sanofi- Synthe labo WO 0242269 1194

Sanofi- Synthe labo WO 0242269 1195

Cell Thera- peutics WO 0268421 1196

Cell Thera- peutics WO 0268421 1197

Sumi- tomo Pharma- ceuti cals EP 248399 1198

Aventis Pharma EP 248734 1199

Sumi- tomo Pharma- ceuti cals EP 248399 1200

Sumi- tomo Pharma- ceuti cals EP 248399 1201

Sumi- tomo Pharma- ceuti cals EP 248399 1202

Sumi- tomo Pharma- ceuti cals EP 248399 1203

Sumi- tomo Pharma- ceuti cals EP 248399 1204

Sumi- tomo Pharma- ceuti cals EP 248399 1205

Sumi- tomo Pharma- ceuti cals EP 248399 1206

Sumi- tomo Pharma- ceuti cals EP 248399 1207

Aventis Pharma EP 248734 1208

Aventis Pharma EP 248734 1209

Aventis Pharma EP 248734 1210

Aventis Pharma EP 248734 1211

Aventis Pharma EP 248734 1212

Aventis Pharma EP 248734 1213

Pfizer AU 8783281 1214

Harbor Branch Found. U.S. Pat. No. 4755529 1215

Roche Bio- science AU 8782540 1216

Roche Bio- science AU 8782540 1217

Roche Bio- science AU 8782540 1218

Roche Bio- science AU 8782540 1219

Novar- tis EP 296110 1220

Novar- tis 1) Lam, C. et al. Antimicrob Agents Chemother 1991, 35(3): 500. AU 8822785 1221

Scher- ing- Plough EP 318214 1222

Novar- tis AU 8822785 1223

Novar- tis AU 8822785 1224

Novar- tis EP 296110 1225

Novar- tis EP 296110 1226

Novar- tis EP 296110 1227

Scher- ing- Plough EP 318214 1228

Leo WO 8910351 1229

Leo WO 8910351 1230

Leo WO 8910351 1231

Leo WO 8910351 1232

Leo WO 8910351 1233

Leo WO 8910351 1234

Leo WO 8910351 1235

Tanabe Seiyaku 1) Ueno, M. et al. Jpn J Pharmacol 1992, 58 (Suppl. 1): Abst O-210. AU 8942368 1236

Green- wich Pharm. AU 9047648 1237

Green- wich Pharm. AU 9047648 1238

Aventis Pharma EP 378456 1239

Aventis Pharma EP 378456 1240

Roche EP 384349 1241

SPA EP 421074 1242

Hitachi Chemi- cal EP 421682 1243

Fuji- sawa 1) Manda, T. et al. Jpn J Pharmacol 1997, 73 (Suppl. 1): Abst P-140. EP 412404 1244

Leo WO 9109841 1245

Hitachi Chemi- cal EP 421682 1246

Hitachi Chemi- cal EP 421682 1247

Hitachi Chemi- cal EP 421682 1248

Hitachi Chemi- cal EP 421682 1249

Hitachi Chemi- cal EP 421682 1250

Hitachi Chemi- cal EP 421682 1251

Hitachi Chemi- cal EP 421682 1252

Hitachi Chemi- cal EP 421682 1253

SPA EP 421074 1254

SPA EP 421074 1255

SPA EP 421074 1256

SPA EP 421074 1257

SPA EP 421074 1258

SPA EP 421074 1259

SPA EP 421074 1260

SPA EP 421074 1261

SPA EP 421074 1262

SPA EP 421074 1263

SPA EP 421074 1264

SPA EP 421074 1265

SPA EP 421074 1266

SPA EP 421074 1267

SPA EP 421074 1268

SPA EP 421074 1269

Leo EP 506794 1270

Fiji- sawa WO 9119708 1271

Harbor Branch Found. U.S. Pat. No. 5091368 1272

Aventis Pharma EP 476658 1273

Merck & Co. EP 480713 1274

Aventis Pharma EP 476658 1275

Aventis Pharma EP 476658 1276

Aventis Pharma EP 476658 1277

Aventis Pharma EP 476658 1278

Aventis Pharma EP 476658 1279

Aventis Pharma EP 476658 1280

Aventis Pharma EP 476658 1281

Aventis Pharma EP 476658 1282

Harbor Branch Found. U.S. Pat. No. 5091368 1283

Merck & Co. EP 480713 1284

Merck & Co. EP 480713 1285

Merck & Co. EP 480713 1286

Merck & Co. EP 480713 1287

Kyowa Hakko Miwa, K. et al. 113th Annu Meet Pharmaceut Soc Jpn (March 29-31, Osaka) 1993, Abst 30CC 13-1. EP 505058 1288

Fuji- sawa Nakamura, K. et al. Chem Pharm Bull 1993, 41(5): 894. AU 8783152 1289

Wyeth U.S. Pat. No. 5312831 1290

Wyeth U.S. Pat. No. 5312831 1291

Wyeth U.S. Pat. No. 5312831 1292

Cell Thera- peutics WO 9416704 1293

Immu- nex 1) Immunex Corporation Press Release 1994, July 21. WO 9506031 1294

Cell Thera- peutics WO 9416704 1295

Cell Thera- peutics WO 9416704 1296

Cell Thera- peutics WO 9416704 1297

Cell Thera- peutics WO 9416704 1298

Cell Thera- peutics WO 9416704 1299

Cell Thera- peutics WO 9416704 1300

Cell Thera- peutics WO 9416704 1301

Abbott GmbH WO 9500493 1302

Glaxo- Smith Kline WO 9504734 1303

Abbott GmbH WO 9500493 1304

Abbott GmbH WO 9500493 1305

Abbott GmbH WO 9500493 1306

Abbott GmbH WO 9500493 1307

Abbott GmbH WO 9500493 1308

Abbott GmbH WO 9500493 1309

Abbott GmbH WO 9500493 1310

Abbott GmbH WO 9500493 1311

Abbott GmbH WO 9500493 1312

Abbott GmbH WO 9500493 1313

Abbott GmbH WO 9500493 1314

Sanofi- Synthe- labo EP 644197 1315

Sanofi- Synthe- labo CA 2125021 1316

Glaxo Smith Kline WO 9504734 1317

Glaxo Smith Kline WO 9504734 1318

Glaxo Smith Kline WO 9504734 1319

Glaxo Smith Kline WO 9504734 1320

Glaxo Smith Kline WO 9504734 1321

Glaxo Smith Kline WO 9504734 1322

Japan Tobac- co JP 95002779 1323

Pharma- cia 1) Gozzi, P. et al. J Pharmacol Exp Ther 1999, 291(1): 199. JP 1995501330 1324

Sanofi- Synthe- labo EP 644197 1325

Sanofi- Synthe- labo EP 644197 1326

Sanofi- Synthe- labo EP 644197 1327

Sanofi- Synthe- labo CA 212021 1328

Sanofi- Synthe- labo CA 212021 1329

Sanofi- Synthe- labo CA 212021 1330

Sanofi- Synthe- labo CA 212021 1331

Sanofi- Synthe- labo CA 212021 1332

Sanofi- Synthe- labo CA 212021 1333

Sanofi- Synthe- labo CA 212021 1334

Lilly WO 9517382 1335

Millen- nium WO 9518610 1336

Aventis Pharma WO 9520578 1337

Cell Thera- peutics WO 9522546 1338

Lilly WO 9517382 1339

Lilly WO 9517382 1340

Lilly WO 9517382 1341

Lilly WO 9517382 1342

Lilly WO 9517382 1343

Lilly WO 9517382 1344

Lilly WO 9517382 1345

Lilly WO 9517382 1346

Lilly WO 9517382 1347

Lilly WO 9517382 1348

Cyto Med WO 9518610 1349

Cyto Med WO 9518610 1350

Cyto Med WO 9518610 1351

Cyto Med WO 9518610 1352

Cyto Med WO 9518610 1353

Cyto Med WO 9518610 1354

Cyto Med WO 9518610 1355

Cyto Med WO 9518610 1356

Cyto Med WO 9518610 1357

Cyto Med WO 9518610 1358

Cyto Med WO 9518610 1359

Cyto Med WO 9518610 1360

Cyto Med WO 9518610 1361

Cyto Med WO 9518610 1362

Cyto Med WO 9518610 1363

Cyto Med WO 9518610 1364

Cyto Med WO 9518610 1365

Cyto Med WO 9518610 1366

Duphar EP 664287 1367

Japan Tobac- co JP 95002779 1368

Japan Tobac- co JP 95002779 1369

Japan Tobac- co JP 95002779 1370

Japan Tobac- co JP 95002779 1371

Japan Tobac- co JP 95002779 1372

Japan Tobac- co JP 95002779 1373

Japan Tobac- co JP 95002779 1374

Japan Tobac- co JP 95002779 1375

Aventis Pharma WO 9520578 1376

Aventis Pharma WO 9520578 1377

Aventis Pharma WO 9520578 1378

Aventis Pharma WO 9520578 1379

Sanofi- Synthe- labo WO 9526958 1380

Cell Thera- peutics WO 9522546 1381

Cell Thera- peutics WO 9522546 1382

Cell Thera- peutics WO 9522546 1383

Cell Thera- peutics WO 9522546 1384

Cell Thera- peutics WO 9522546 1385

Cell Thera- peutics WO 9522546 1386

Duphar EP 664287 1387

Duphar EP 664287 1388

Duphar EP 664287 1389

Duphar EP 664287 1390

Duphar EP 664287 1391

Duphar EP 664287 1392

Sanofi- Synthe- labo WO 9526958 1393

Sanofi- Synthe- labo WO 9526958 1394

Daikin EP 711766 1395

Sanofi- Synthe- labo WO 9533751 1396

Ajino- moto U.S. Pat. No. 5464918 1397

Ajino- moto U.S. Pat. No. 5464918 1398

Ajino- moto U.S. Pat. No. 5464918 1399

Ajino- moto U.S. Pat. No. 5464918 1400

Sanofi- Synthe- labo WO 9533751 1401

Sanofi- Synthe- labo WO 9533751 1402

Sanofi- Synthe- labo WO 9533751 1403

Sanofi- Synthe- labo WO 9533751 1404

Sanofi- Synthe- labo WO 9533751 1405

Sanofi- Synthe- labo WO 9533751 1406

Daikin EP 711766 1407

Daikin EP 711766 1408

Daikin EP 711766 1409

Daikin EP 711766 1410

Micro- bial Chem- istry Re- search Founda- tion JP 96176157 1411

Tanabe WO 9640641 1412

Tanabe WO 9640641 1413

Daiichi Pharma- ceutical 1) Kawagoe, K. et al. AFMC Int Med Chem Symp (Sept 3-8, Tokyo) 1995, Abst P13M183. JP 97059236 1414

Vertex WO 9722618 1415

Vertex WO 9722618 1416

Vertex WO 9722618 1417

Vertex WO 9722618 1418

Vertex WO 9722618 1419

Vertex WO 9722618 1420

Astra Zeneca WO 9731023 1421

Univer- sity of Penn- sylvania WO 9733603 1422

Astra Zeneca WO 9731023 1423

Astra Zeneca WO 9731023 1424

Astra Zeneca WO 9731023 1425

Astra Zeneca WO 9731023 1426

Leo WO 9737972 1427

Taisho JP 97194476 1428

Leo WO 9737972 1429

Leo WO 9737972 1430

Leo WO 9737972 1431

Leo WO 9737972 1432

Leo WO 9737972 1433

Glaxo- Smith Kline WO 9743250 1434

Pharm- acia WO 9745409 1435

Pharm- acia WO 9745409 1436

Pharm- acia WO 9745409 1437

Pharm- acia WO 9745409 1438

LEK EP 477912 1439

Astra Zeneca WO 9828275 1440

Astra Zeneca WO 9828275 1441

Astra Zeneca WO 9828270 1442

Astra Zeneca WO 9828270 1443

Astra Zeneca WO 9828270 1444

Astra Zeneca WO 9828270 1445

Astra Zeneca WO 9828270 1446

Astra Zeneca WO 9828270 1447

Astra Zeneca WO 9828270 1448

Astra Zeneca WO 9828270 1449

Astra Zeneca WO 9828270 1450

Astra Zeneca WO 9828270 1451

Astra Zeneca WO 9828270 1452

Astra Zeneca WO 9828270 1453

Astra Zeneca WO 9828270 1454

Astra Zeneca WO 9828270 1455

Astra Zeneca WO 9828270 1456

Astra Zeneca WO 9828270 1457

Astra Zeneca WO 9828270 1458

Astra Zeneca WO 9828270 1459

Daiichi Pharma- ceutical Koiwa, T. et al. J Antibiot 1999, 52(2): 198. 1460

Bristol- Myers Squibb WO 0244181 1461

Bristol- Myers Squibb WO 0244181 1462

Bristol- Myers Squibb WO 0244181 1463

Bristol- Myers Squibb WO 0244181 1464

Bristol- Myers Squibb WO 0244181 1465

Bristol- Myers Squibb WO 0244181 1466

Bristol- Myers Squibb WO 0244181 1467

Bristol- Myers Squibb WO 0244181 1468

Bristol- Myers Squibb WO 0244181 1469

Gruen- enthal WO 0290317 

1. An isolated polypeptide of the SARS virus.
 2. The polypeptide of claim 1, wherein the polypeptide is a Spike (S) polypeptide, an Env (E) polypeptide, a Membrane (M) polypeptide, a hemagglutinin-esterase polypeptide (HE), a nucleocapsid (N) polypeptide, a ORF1a polypeptide, a ORF1ab polypeptide, a proteolytic fragment of a ORF1a polypeptide, or a proteolytic fragment of a ORF1ab polypeptide.
 3. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 6039, 7232, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050 or
 6052. 4. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence having >75% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633 &
 11636. 5. The polypeptide of claim 1, wherein the polypeptide comprises a fragment of at least 10 consecutive amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11552, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633 &
 11636. 6. A polypeptide comprising an amino acid sequence having >80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11552, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633 &
 11636. 7. A polypeptide comprising an amino acid sequence that comprises a fragment of at least 10 consecutive amino acids of an amino acid sequence selected from the group consisting SEQ ID NOS: 6042, 6043, 6044, 6045, 6046, 6047, 6048, 6049, 6050, 6052, 9766, 9767, 9768, 9769, 9770, 9771, 9772, 9773, 9774, 9775, 9776, 9777, 9778, 9779, 9997, 9998, 10149, 10316, 10338, 10339, 10340, 10341, 10342, 10532, 10533, 10571, 10572, 10573, 10574, 10575, 10576, 10577, 10578, 10579, 11552, 11561, 11562, 11618, 11619, 11620, 11627, 11630, 11633 &
 11636. 8. A polypeptide comprising an amino acid sequence having >80% sequence identity to SEQ ID NO: 6042, and/or comprising an amino acid sequence that comprises a fragment of at least 10 consecutive amino acids of SEQ ID NO: 6042, wherein the polypeptide is in the form of a trimer.
 9. Nucleic acid encoding the polypeptide of any one of claims 1 to
 8. 10. Nucleic acid according to claim 9, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 7191, 7273, 7275, 7277, 7279, 7281, 7283, 7285, 7287, 7289, 7291, 7292, 7293, 9968, 10066, 10084, 10299, 10505, 11323, 11563, 11639 &
 11640. 11. A polynucleotide comprising a nucleotide sequence having >80% sequence identity to the nucleic acid of claim 9 or claim
 10. 12. A polynucleotide comprising a fragment of at least 10 consecutive nucleotides of the nucleic acid of claim 9 or claim
 10. 13. Antibody that recognizes the polypeptide of any one of claim 1 to
 8. 14. The antibody of claim 13, wherein said antibody recognizes the polypeptide comprising the amino acid sequence of SEQ ID NO: 6042 or a fragment thereof.
 15. The antibody of claim 14, wherein said antibody recognizes the polypeptide comprising the amino acid sequence of SEQ ID NO: 6042 or a fragment thereof in trimeric form.
 16. The antibody of claim 13, wherein the antibody is a monoclonal antibody.
 17. The antibody of claim 13, wherein the antibody is a human antibody.
 18. An immunoassay for detecting a SARS virus antigen in a sample, comprising the step of contacting the sample with the antibody of any one of claims 13 to
 17. 19. An immunoassay for detecting an antibody against a SARS virus antigen in a sample, comprising the step of contacting the sample with the polypeptide of any one of claims 1 to
 8. 20. A method of detecting an antibody against a SARS virus antigen in a sample comprising contacting said sample with the polypeptide of any one of claims 1 to 8, under conditions suitable for binding said polypeptide to said antibody, if present, and detecting the binding of said polypeptide to said antibody.
 21. A method for detecting a SARS virus antigen in a sample comprising contacting said sample with the antibody of any one of claims 13 to 17, under conditions suitable for binding said antibody to said antigen, if present, and detecting the binding of said antibody to said antigen.
 22. A vaccine for the treatment or prevention of severe acute respiratory syndrome (SARS), comprising an inactivated SARS virus, a killed SARS virus, an attenuated SARS virus, a split SARS virus preparation, or at least one purified SARS virus antigens.
 23. The vaccine of claim 22, comprising a purified polypeptide according to any one of claims 1 to
 8. 24. The vaccine of claim 22 or claim 23, wherein the antigen is a purified SARS virus antigen in the form of a VLP.
 25. The vaccine of any one of claims 22 to 24, further comprising an adjuvant.
 26. The vaccine of claim 25, wherein the adjuvant is an aluminium salt or is MF59.
 27. The vaccine of any one of claims 22 to 26, comprising more than one SARS virus antigen.
 28. The vaccine of claim 27, wherein the antigens are selected from S, E, N and M.
 29. The vaccine of claim 22, comprising an inactivated SARS virus.
 30. The vaccine of claim 29, wherein said virus is inactivated by chemical or physical means.
 31. The vaccine of claim 30, wherein said inactivation comprises treatment of the virus with an effective amount of one or more of the following agents selected from the group consisting of detergents, formaldehyde, formalin, β-propriolactone, and UV light.
 32. The vaccine of claim 30, wherein said inactivation comprises treatment of the virus with an effective amount of one or more of the following agents selected from the group consisting of methylene blue, psoralen and carboxyfullerene (C60).
 33. The vaccine of claim 30, wherein said inactivation comprises treatment of the virus with an effective amount of one or more of the following agents selected from the group consisting of binary ethylamine, acetyl ethyleneimine and gamma irradiation.
 34. The vaccine of claim 31, wherein said inactivation comprises treatment with β-propriolactone.
 35. The vaccine of claim 34, wherein said β-propriolactone is used at a concentration of 0.01 to 0.5%.
 36. The vaccine of claim 34, wherein said β-propriolactone is used at a concentration of 0.5 to 0.2%.
 37. The vaccine of claim 34, wherein said β-propriolactone is used at a concentration of 0.025 to 0.1%.
 38. A method of inactivating SARS virus comprising exposing the virus to an inactivation agent for 12 to 24 hours at refrigeration temperatures followed hydrolysis of any residual inactivating agent by elevating the temperature for three hours.
 39. The method of claim 38, wherein the inactivation agent is β-propriolactone.
 40. The method of claim 38, wherein the refrigeration temperature is between 0° C. and 8° C.
 41. The method of claim 38, wherein the elevated temperature is between 33° C. and 41° C.
 42. A method for making an inactivated SARS vaccine comprising: a. innoculating a mammalian cell culture with SARS virus; b. cultivating the infected cells; c. harvesting SARS virus containing supernatant; d. inactivating the SARS virus; and e. purifying the inactivated SARS virus.
 43. The method of claim 42, wherein said mammalian cell culture is derived from one or more of the cell types selected from the group consisting of fibroblast cells, endothelial cells, hepatocytes, keratinocytes, immune cells, mammary cells, smooth muscle cells, melanocyte cells, neural cells, prostate cells, renal cells, skeletal cells, liver cells, retinoblast cells and stromal cells.
 44. The method of claim 42, wherein said mammalian cell culture is derived from a cell culture selected from the group consisting of human cells, non-human primate cells, HeLa cells, human diploid cells, fetal rhesus lung cells, human embryonic kidney cells, VERO cells, horse cells, cow cells, sheep cells, dog cells, cat cells or rodent cells.
 45. The method of claim 42, wherein said mammalian cell culture is derived from VERO cells or fetal rhesus kidney cells.
 46. The method of claim 42, wherein said mammalian cells are cultured in serum free media.
 47. The method of claim 42, wherein said mammalian cells are cultured in protein free media.
 48. The method of claim 42, wherein said inoculating step comprising absorbing the SARS virus onto the cell culture for 60 to 300 minutes.
 49. The method of claim 42, wherein said inoculating step is conducted at 25° C. to 40° C.
 50. The method of claim 42, wherein said purification step comprises one or more of the treatments selected from the group consisting of gradient centrifugation, ultracentrifugation, continuous-flow ultracentrifugation, chromatography, polyethylene glycol precipitation, and ammonium sulfate precipitation.
 51. The method of claim 42, wherein said purification step comprises one or more of the treatments selected from the group consisting of ultrafiltration and dialfiltration.
 52. The method of claim 50, wherein said chromatography treatment includes one or more of the chromatography treatments selected from the group consisting of ion exchange chromatography, size exclusion chromatography, and liquid affinity chromatography.
 53. The method of claim 52, wherein said chromatography treatment includes use of one more chromatographic resins selected from the group consisting of an an anionic resin and a cationic resin.
 54. The method of claim 52, wherein the ion exchange chromatography treatment includes a first step using a strong anion exchange resin and a second step using a strong cation exchange resin.
 55. The method of claim 50, wherein said gradient centrifugation purification step comprises density gradient centrifugation.
 56. The method of claim 42, wherein said purification step comprises a first step of chromatography purification and a second step of gradient centrifugation.
 57. The method of claim 56, wherein said first chromatography purification step comprises liquid affinity chromatography.
 58. The method of claim 56, wherein said second gradient centrifugation step comprises density gradient centrifugation.
 59. A single-stranded oligonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 21-6020, 6076-6568, 6586-6587, 7292-7301, 7325-7328, 7332-7352, 7353-7385, 10235-10298, 10352-10504, 10580-11322 and 11325-11551.
 60. A single-stranded oligonucleotide comprising the complement of the oligonucleotide of claim
 59. 61. The oligonucleotide of claim 59 or claim 60, comprising 10-30 nucleotides.
 62. The oligonucleotide of claim 61, comprising the nucleotide sequence of SEQ ID NO: 7292, SEQ ID NO: 7293, the complement of SEQ ID NO: 7292 or the complement of SEQ ID NO:
 7293. 63. A kit comprising primers for amplifying a template sequence contained within a SARS virus nucleic acid target, the kit comprising a first primer and a second primer, wherein the first primer comprises a sequence substantially complementary to a portion of said template sequence and the second primer comprises a sequence substantially complementary to a portion of the complement of said template sequence, wherein the sequences within said primers which have substantial complementarity define the termini of the template sequence to be amplified.
 64. The kit of claim 63, wherein the template sequence is contained within SEQ ID NO: 1 and/or SEQ ID NO:
 2. 65. The kit of claim 63 or claim 64, wherein the first primer comprises a fragment of 8 or more nucleotides of SEQ ID NO: 1, and the second primer comprises a fragment of 8 or more nucleotides of the complement of SEQ ID NO:
 1. 66. The kit of claim 63 or claim 64, wherein the first primer comprises a fragment of 8 or more nucleotides of SEQ ID NO: 2, and the second primer comprises a fragment of 8 or more nucleotides of the complement of SEQ ID NO:
 2. 67. The kit of claim 63, wherein the first primer is an oligonucleotide according to any one of claims 59 to 62 and the second primer is an oligonucleotide according to any of claims 59 to
 62. 68. The kit of any one of claims 63 to 67, further comprising a labeled probe that comprises either a fragment of 8 or more nucleotides of SEQ ID NO: 1 and/or SEQ ID NO: 2, or the complement of said fragment, which fragment is located within the template sequence.
 69. The kit of any one of claims 63 to 68, wherein the first primer and/or the second primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 21-6020, 6076-6568, 6586-6587, 7292-7301, 7325-7328, 7332-7352, 7353-7385, 10235-10298, 10352-10504, 10580-11322 and 11325-11551.
 70. The kit of any one of claims 63 to 68, wherein the first primer and/or the second primer comprises the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 21-6020, 6076-6568, 6586-6587, 7292-7301, 7325-7328, 7332-7352, 7353-7385, 10235-10298, 10352-10504, 10580-11322 and 11325-11551.
 71. A method of detecting the presence of SARS virus in a sample comprising providing a sample suspected of containing a SARS virus nucleic acid target, amplifying a template sequence contained within said SARS virus nucleic acid target with the kit of any one of claims 63 to 70, and detecting the amplified template sequence, wherein the presence of the amplified template sequence indicates the presence of SARS virus in said sample.
 72. The method of claim 71, wherein said amplifying is accomplished using polymerase chain reaction, transcription mediated amplification, reverse transcription PCR, ligase chain reaction, strand displacement amplification or nucleic acid sequence-based amplification.
 73. A double-stranded RNA molecule with a length from about 10 to about 30 nucleotides which is able to inactivate the SARS coronavirus in a mammalian cell.
 74. The double-stranded RNA of claim 73, wherein the sequence of one of the strands is at least 90% identical to a target sequence, wherein the target sequence is a fragment of SEQ ID NO: 1 and/or SEQ ID NO:
 2. 75. The double-stranded RNA of claim 73 or claim 74, wherein the target sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS: 7292, 7293, 7294, 7295, 7296, 7297, 7298, 7299, 7300 and
 7301. 76. The double-stranded RNA of any one of claims 73 to 75, comprising at least one modified nucleotide.
 77. A method for treating a patient suffering from SARS, comprising: administering to the patient a therapeutically effective dose of a molecule of less than 1000 g/mol.
 78. The method of claim 77, wherein the molecule has an aromatic region and greater than one heteroatom selected from O, S, or N.
 79. A method for treating a patient suffering from SARS, comprising: administering to the patient a therapeutically effective dose of a compound selected from: a nucleoside analog, a peptoid, an oligopeptide, a polypeptide a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA dependent RNA polymerase.
 80. A method for treating a patient suffering from SARS, comprising: administering to the patient a steroidal anti-inflammatory drug in combination with at least one antiviral compound.
 81. A method for treating a patient suffering from SARS, comprising: administering to the patient a therapeutically effective dose of a compound selected from: acyclovir, gancyclovir, vidarabidine, foscamet, cidofovir, amantidine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, an antiviral compound listed in Table 1; an antiviral compound listed in Table 2; or an interferon.
 82. The method of claim 81, wherein the interferon is an interferon-α or an interferon-β.
 83. The method of any one of claims 77 to 82, wherein the molecule or compound is delivered by inhalation.
 84. A method of identifying a therapeutically active agent comprising the steps of: (a) contacting a therapeutically active agent with a cell infected with the SARS virus; (b) measuring attenuation of a SARS related enzyme.
 85. A viral vector or particle for in vivo delivery of a nucleic acid of claim 9 or claim
 10. 86. The viral vector of claim 85, wherein the vector is an adenovirus vector, a poxvirus vector or an alphavirus vector.
 87. An alphavirus replicon particle comprising one or more SARS viral antigens.
 88. The replicon particle of claim 87, wherein said SARS viral antigen is a spike protein.
 89. The replicon particle of claim 87, wherein said particle comprises a replicon derived from Venezuelan Equine Encephalitis (VEE) and further comprises an envelope derived from Sindbus virus (SIN) or Semliki Forest Virus (SFV).
 90. A vaccine comprising one or more SARS virus antigens and one or more respiratory virus antigens.
 91. The vaccine of claim 90, wherein said respiratory virus antigens are selected from the group consisting of influenza virus, human rhinovirus (HRV), parainfluenza virus (PIV), respiratory syncytial virus (RSV), adenovirus, metapneumovirus, and rhinovirus.
 92. The vaccine of claim 91, wherein said respiratory virus antigen is from influenza virus.
 93. The vaccine of claim 90, wherein said respiratory virus antigen is from a coronavirus other than the SARS virus.
 94. A polypeptide comprising an immunogenic, surface exposed fragment of the amino acid sequence SEQ ID NO:
 6042. 95. The polypeptide of claim 94, wherein said fragment does not include the last 50 amino acids of the C-terminus of SEQ ID NO:
 6042. 96. The polypeptide of claim 94, wherein said fragment does not include a transdomain region of SEQ ID NO:
 6042. 97. The polypeptide of claim 94, wherein said fragment does not include a C-terminus cytoplasmic domain of SEQ ID NO:
 6042. 98. The polypeptide of claim 94, wherein said fragment does not include a N-terminus signal sequence.
 99. An isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 9968 and
 10066. 100. The polynucleotide of claim 99, wherein the polynucleotide comprising a nucleic acid sequence having >80% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 9968 and
 10066. 101. An isolated polynucleotide comprising a fragment of at least 15 consecutive nucleic acids of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 9968 and 10066 and wherein said fragment does not consist entirely of SEQ ID NO:
 10033. 102. An isolated polypeptide comprising an amino acid sequence encoded by any one of claims 99-101.
 103. The polypeptide of claim 102, comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 9969-10032, 10067, and
 10015. 104. The polypeptide of claim 103, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOS: 9997, 9998 and
 10015. 105. An expression construct for recombinant expression of a SARS virus spike protein wherein said construct comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 6578-6583.
 106. A mammalian cell line stably expressing a SARS viral antigen.
 107. The cell line of claim 106, wherein said cell line is a Chinese Hamster Ovary (CHO) cell.
 108. The cell line of claim 106, wherein the SARS viral antigen is a spike protein or fragment thereof.
 109. The cell line of claim 106, wherein the spike protein is truncated to remove the transmembrane sequence.
 110. A method of identifying a therapeutically active agent comprising the steps of: (a) contacting a therapeutically active agent with a buffer comprising SARS enzyme; and (b) measuring attenuation of the SARS enzyme.
 111. The method of claim 110 wherein the SARS enzyme is a SARS protease.
 112. The method of claim 111 wherein the buffer further comprises a peptide with a SARS protease cleave site.
 113. The method of claim 110 wherein the measurement is made by the measurement of fluorescence.
 114. A vaccine of one of claims 22 to 37, and 90 to 93 further comprising an adjuvant.
 115. The vaccine of claim 114 wherein the adjuvant is a SMIP.
 116. The vaccine of claim 115 wherein the SMIP compound is selected from the group consisting of an acylpiperazine, a tryptanthrin, an indoledione, a tetrahydroisoquinoline, a benzocyclodione, an amino azavinyl compound, a thiosemicarbazone, a lactam, an aminobenzimidazole quinolinone, a hydropthalamide, a benzophenone, an isoxazole, a sterol, a quinazolinone, a pyrole, an anthraquinone, a quinoxaline, a triazine, an benzazole, and a pyrazolopyrimidine, or a pharmaceutically acceptable salt, ester, or prodrug thereof.
 117. A method of vaccinating a subject comprising administering a vaccine of one of claims 22 to 37, and 90 to
 93. 118. The method of claim 117 further comprising administering a SMIP.
 119. A method for treating a patient of one of claims 77 to 82 further comprising administering at least one SMEP compound.
 120. A method for treating a patient of one of claims 77 to 82 further comprising administering at least one SMIS compound. 